The last few years have seen tremendous advances in CRISPR-mediated genome editing. Great efforts have been made to improve the efficiency, specificity, editing window, and targeting scope of CRISPR/Cas9-mediated transgene knock-in and gene correction. In this article, we comprehensively review recent progress in CRISPR-based strategies for targeted transgene knock-in and gene correction in both homology-dependent and homology-independent approaches. We cover homology-directed repair (HDR), synthesis-dependent strand annealing (SDSA), microhomology-mediated end joining (MMEJ), and homology-mediated end joining (HMEJ) pathways for a homology-dependent strategy and alternative DNA repair pathways such as non-homologous end joining (NHEJ), base excision repair (BER), and mismatch repair (MMR) for a homology-independent strategy. We also discuss base editing and prime editing that enable direct conversion of nucleotides in genomic DNA without damaging the DNA or requiring donor DNA. Notably, we illustrate the key mechanisms and design principles for each strategy, providing design guidelines for multiplex, flexible, scarless gene insertion and replacement at high efficiency and specificity. In addition, we highlight next-generation base editors that provide higher editing efficiency, fewer undesired by-products, and broader targeting scope. Using CRISPR, a specific genetic variant associated with human disease can be introduced into the genome, or a mutation in the genome can be replaced with a wild-type allele, at will by design in cells and organisms, respectively, for gene knock-in or gene correction12. The CRISPR-modified cellular and animal models then can be studied to uncover mechanisms underlying human disease and for drug discovery15. Therefore, CRISPR holds tremendous translational potential. CRISPR technology has been explored for in vivo gene therapy to treat a wide range of human hereditary diseases18 as well as ex vivo gene therapy to treat blood disorders, cancers, and immune-related diseases by genetically modifying the patient’s cells outside their body23. The last few years have seen enormous advances to improve the performance and maximize the potential of CRISPR-mediated gene insertion and replacement for disease modeling and gene therapeutics.Clustered regularly interspaced short palindromic repeats (CRISPR) has revolutionized the field of transgenic research and human gene therapy27, single-stranded donor oligonucleotide (ssODN)30, linear double-stranded polymerase chain reaction (PCR) fragments32, or the homologous sequences of the intact sister chromatid33. Depending on the forms of repair template and CRISPR system used, homology-mediated gene insertion and replacement are carried out via specific DNA repair pathways such as homology-directed repair (HDR)33, synthesis-dependent strand annealing (SDSA)34, microhomology-mediated end joining (MMEJ)36, and homology-mediated end joining (HMEJ)39 pathways. In homology-independent approaches, transgene knock-in and gene correction are achieved with CRISPR/Cas9 in the absence of DNA donor repair templates. In this case, the error-prone non-homology end joining (NHEJ) repair pathway is commonly used to repair CRISPR-mediated double-stranded breaks and to incorporate a mutagenic sequence into the genome41.The strategies for transgene knock-in and gene correction are generally classified into homology-dependent and homology-independent. Homology-directed repair approaches require the presence of both CRISPR/Cas9 and a DNA donor repair template. The repair template can be in the form of circular double-stranded plasmid DNA45 and primer editing46 are used to perform gene correction and replacement by directly converting nucleotides in genomic DNA without damaging the DNA or requiring donor DNA. Various base-editor variants have also recently been engineered to provide higher editing efficiency, fewer undesired by-products, and broader targeting scope. Simultaneous substitution of multiple nucleotides has been achieved by fusing Cas9 nickase (Cas9n) to a DNA polymerase47 or a reverse transcriptase (RT)46. To avoid permanent deleterious effects caused by unanticipated mutagenesis and complex chromosomal rearrangements to genomic DNA, CRISPR is employed to edit the endogenous RNA transcripts containing pathogenic mutations50.As alternatives, base editingGiven the explosive popularity and rapid evolution of CRISPR technology in transgenic research and human gene therapy, the purpose of this review is to provide a comprehensive summary on various, recently developed CRISPR-based strategies for controlled and desired transgene knock-in and gene correction as a quick reference for those who are new to this fast-moving field. We illustrate and highlight the mechanisms of action and key concepts for each transgene knock-in and gene correction strategy. We elaborate design guidelines for scarless gene insertion and replacement at high efficiency and specificity. We also critically discuss the applications and pros and cons of each strategy. Finally, we briefly discuss how engineered CRISPR variants and alternate donor template designs have improved the efficiency, specificity, editing window, and targeting scope of transgene knock-in and gene correction.51. To introduce an exonic mutation, CRISPR/Cas9 ribonucleoprotein (RNP) complex is co-delivered with the plasmid DNA donor containing two homology arms (~800 bp each arm) flanking the mutated sequence into the cell . It allows desired and controlled genetic modifications in the genome. This HDR approach requires the presence of a DNA repair template, usually in the form of circular plasmid DNA. However, HDR activity can be improved using the linearized plasmid with the short 5′ backbone overhangthe cell . As an o26. After the two-step editing process, the edited cells with no fluorescent protein expression are flow-sorted and single-cell-expanded for characterization. Alternatively, marker-free genome editing can be achieved by using a highly efficient HDR approach that was developed recently54. This approach uses electroporation to deliver adeno-associated virus (AAV) 6-mediated DNA repair template and Cas9 protein complexed with chemically modified sgRNA. As an alternative, the repair template can be delivered as an ssDNA donor. As the majority of cells are successfully edited, no selection marker  is required to screen a large number of clones to identify biallelic edited clones. Therefore, this scarless genome-editing approach is particularly useful for therapeutic applications in hard-to-transfect cells such as human pluripotent stem cells54, adult stem cells55, and primary immune cells56.For introducing non-coding variants within the regulatory elements , a clean mutational knock-in or scarless genome-editing approach is recommended. For example, the fluorescent marker gene at the intronic region is removed without the addition of any exogenous sequence by designing the single guide RNA (sgRNA) to target the fluorescent marker gene . Upon cl60, efficient site-specific gene correction of both alleles is needed. Moreover, a single-allele perturbation might not be sufficient to cause significant functional alteration of a given gene, especially for disease-associated genetic variants such as non-synonymous SNPs. Thus, transgenic cells with biallelic gene manipulation are usually preferred over monoallelic, even for SNPs in a heterozygous form, to be able to detect the functional impact of the variants on the activity and molecular phenotypes of the targeted gene product. However, owing to the inefficient HDR events, it is often technically more challenging to screen transgenic cells with targeted mutational knock-in at both alleles than monoallelic gene editing. Therefore, a CRISPR/Cas9-mediated biallelic genome targeting with a dual surrogate reporter-integrated donor system was recently developed to facilitate the selection and enrichment of biallelic-modified cells61. This approach uses a CRISPR/Cas9-mediated homologous recombination system and two different sets of reporter transgene  is first inserted into one of the wild-type alleles in the genome. The insertion is done using CRISPR/Cas9-mediated homologous recombination. Expressions of CRISPR/Cas9 then trigger another round of homologous recombination in the cell to modify the remaining wild-type alleles using homology sequences from the previously modified allele. Eventually, all of the wild-type alleles are converted into modified alleles. This strategy allows self-propagation of the desired trait, super-Mendelian inheritance of a transgene, and genetic modifications of specific populations or entire species.Gene correction has also been achieved via mitotic recombination using the endogenous wild-type allele on the homologous chromosome as a template 33. In th66. Compared with the surrogate reporter-integrated donor system61, the HDR-USR system functions by itself in an episomal manner66. Because the HDR-USR surrogate vector is not integrated into the genome, it allows scarless genome editing without introducing insertional mutagenesis and unwanted exogenous sequences to the genome. Therefore, this HDR-USR system is particularly useful for knock-in of non-coding variants to study functional regulatory elements. It also can be employed to knock-in protein-coding variants in exons. The HDR-USR system contains a truncated puromycin-resistant gene ant gene . Puromyc67. Importantly, 5′ modification of the donors can tackle the problem associated with the integration of multimers due to joining together the multiple templates end to end, thereby enabling only one whole copy of the desired DNA to be inserted into the genome. This strategy also enables DNA to be edited in a more controlled way and promotes precise gene knock-in. Cas9 RNP complexes and 5′-modified linear double-stranded DNA donors with short homology arms (~50 bp) have also successfully been used to enable efficient gene knock-in68. This 5′-end modification of donor does not require long homology arms  and helps to reduce the background signal from the DNA repair templates. Modulation of targeting vectors to provide 3′ overhangs at both ends could also enhance HDR frequency by facilitating the assembly of proteins involved in strand invasion and new DNA synthesis in HDR69.It was recently shown that 5′ modification to the PCR-generated long dsDNA donors (~400 bp) strongly enhances HDR events 67. Impor2/M cell cycle phase70. Alternatively, Cas9 expression is synchronized in S/G2/M by controlled timing of CRISPR/Cas9 RNP complex delivery71 or using a fusion of Cas9 and the N-terminal region of human Geminin25. HDR has also been successfully activated in quiescent stem cells and primary cells through controlled cycling and quiescence72. In this case, these quiescent cells are induced to briefly enter the cell cycle for HDR-based gene editing using the timed administration of a small-molecule cocktail . After gene editing, these HDR-edited cells are induced back into quiescence using another combination of small molecules . A modified version of Cas9 has also been employed to increase the HDR/NHEJ ratio by limiting the Cas9 nuclease activity in the G1 phase of the cell cycle73. HDR efficiency has also been improved by using small molecules that directly modulate HDR pathways, such as RS-1 (RAD51 activator)75, L755507 (β3-adrenergic receptor agonist)76, and nocodazole (a G2/M phase synchronizer)27. Ectopic co-expression of RAD52 and dn53BP1 could improve HDR efficacy without altering Cas9 off-target activity77. In addition, overexpression of an engineered RAD18 variant (e18) promotes HDR by suppressing the localization of the NHEJ-promoting factor 53BP1 to double-strand breaks78. To further promote HDR events, small molecules are used to inhibit the NHEJ activity by disrupting a key enzyme in the NHEJ pathway, such as DNA ligase IV79, KU7080, and 53BP181. In addition, attenuation of histone deacetylase 1 (HDAC1) and HDAC2 activities was recently shown to facilitate Cas9 access and binding to the targeted DNA, thereby enhancing HDR events and gene knock-in efficiency82. As such, a desired editing outcome can be genetically manipulated or chemically induced by altering the choice of DNA double-strand break repair to favor a specific DNA repair pathway83. For example, small molecules  and factors (genes) identified in high-throughput screens can be used to enhance or inhibit a specific DNA repair pathway for intended genome editing. To realize such applications, many other unknown small molecules and factors that potentially target the DNA damage response can be screened using a recently developed scalable CRISPR/Cas9-based fluorescent reporter assay to assess their influence on DNA double-strand break repair choice84. The underlying mechanisms by which these small molecules and factors regulate double-strand break repair then can be evaluated.Gene knock-in and gene correction efficiency can be increased by enhancing HDR efficiency. HDR events are increased by synchronizing and enriching the cells in the G85. Directing CtIP-mediated homologous recombination to the CRISPR/Cas9 cutting site can minimize a global effect following DNA repair. On the other hand, fusions of Cas9 to DN1S (dominant-negative mutant of 53BP1) enhance HDR and precise gene knock-in by inhibiting NHEJ events specifically at Cas9 cleavage sites86. The use of this Cas9-DN1S fusion protein can circumvent the issue associated with the unwanted effects of global NHEJ inhibition. Another strategy to improve HDR efficiency is to covalently link the DNA repair templates to CRISPR/Cas9 complexes89. For example, to shuttle the HDR template to the nucleus, truncated Cas9 target sequences (16 bp) are added to the ends of the template for interacting with Cas9 RNPs , Cas9 RNPs along with the associated HDR template can be transported together into the cell nucleus. To further enhance HDR efficiency, multiple copies of DNA repair templates are conjugated to multiple sites on a Cas9 via the conjugation adaptor89. Each conjugation adaptor on Cas9 bears the complementary sequence to the DNA repair template and thus multiple conjugation adaptors enable multivalent display of DNA repair template on Cas9. These conjugation strategies ensure the uptake and co-localization of both repair template and Cas9–sgRNA RNP complex in an individual cell88.However, the use of small molecules or disruption of NHEJ and histone modifier genes can cause genome-wide changes in gene regulation and expression. Therefore, Cas9 was recently fused to CtIP, a key protein in early steps of homologous recombination, to stimulate precise transgene integration by HDRas9 RNPs 90. Becau91. Deep learning has helped to predict editing products and genotypes such as on-target mutagenesis and off-target activities upon CRISPR-mediated gene correction of pathogenic variants in human cells96. In addition to aid designing an optimal DNA sequence, deep learning can also improve editing activity of CRISPR by taking into account chromatin accessibility and epigenetic features98.Machine learning has been used to further improve HDR efficiency by designing the optimal targeting strategy and repair template30 and linear double-stranded DNA PCR fragments31 enable efficient gene knock-in via a synthesis-dependent strand annealing (SDSA)-like DNA repair mechanism. Compared with HDR-mediated knock-in using dsDNA plasmid template (>500 bp each homology arm), ssODN (50–80 bp each homology arm) and PCR fragment (~35 bp homology arms) provide higher efficiency for knock-in of small DNA modifications 31. Given their high efficiency of a point mutation knock-in, a fluorescent tag is not required for the PCR fragment or ssODN donor. Several mechanistic models have been proposed to explain how a point mutation is introduced to the genome through DNA repair machinery mediated by a short single-stranded DNA oligonucleotide and CRISPR/Cas9 system. Two promising models are excision and corrective therapy (EXACT)99 and single-stranded template repair (SSTR)100 models. EXACT uses ssODNs to serve as both a bandage and a template for gene correction, whereas SSTR uses the Fanconi anemia DNA repair pathway for targeted mutagenesis.ssODN105. In the case of single-agent gene editing, point mutations and base lesions are repaired by ssODNs in the absence of the CRISPR RNP complex106. This ssODN-mediated DNA repair machinery can be enhanced by stalling of replication forks and synchronizing the cells in the early S cell cycle phase during ssODN exposure107 or using anticancer drugs to induce double-strand DNA breakage109. Subsequently, CRISPR/Cas9 RNP complex has been used to improve point mutation repair directed by a short ssODN through induction of double-stranded breaks at the target genomic loci99. Nevertheless, the mechanism of action of DNA repair directed by an ssODN as the sole gene-editing agent is generally similar to that of ssODN and CRISPR/Cas9 working in concert. Interestingly, single-nick-induced gene editing using ssODN and Cas9n generates conversion tracts biased either mostly unidirectional or bidirectional depending on the relative strandedness of the ssODN and the nick110. In contrast, the unidirectional conversion pathway is preferentially used upon CRISPR/Cas9-induced double-strand DNA breaks. A strand bias in ssODN-mediated gene editing could be due to different accessibility of the DNA strands during DNA replication, irrespective of transcriptional status111. The sequences surrounding the targeted nucleotide also influence strand bias, while occupancy by transcriptional complexes alone does not dictate strand bias112. Notably, unmodified ssODNs are the most efficient in gene correction, while the addition of chemical modifications to the ssODN did not further improve its efficacy111.The mechanism of gene repair directed by the ssODNs was elucidated in detail well before the CRISPR era1 phase of the cell cycle  donor bearing two homology arms (60–300 bp each) along with CRISPR/Cas9 to integrate a long transgene into the genome119.Silent mutations are usually introduced into the PAM sequence or the sgRNA seed sequence of the donor template to block further Cas9 targeting and recutting after undergoing HDR . This call cycle 113. Gem ance HDR . Because121, Cas9n mutant is used to nick single-stranded DNA for seamless genome editing29. DNA nicks are repaired by the alternative HDR pathway , especially when canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated122. Studies have shown that Cas9 D10A  is more potent in mediating HDR than Cas9 H840A 29. For example, Cas9n is fused to hRad51 mutants to improve HDR efficiency while minimizing undesired consequences of double-stranded DNA breaks and off-target mutagenesis  is used to induce a staggered double-strand DNA breaks with overhangs at the target locus by nicking both DNA strands simultaneously  to align the broken strands before joining129. For example, CRISPR/Cas9 is employed to induce a DNA double-stranded break near the center of a disease-causing microduplication  was also recently used to enhance MMEJ-mediated knock-in132. Exo1 facilitates end resection of double-strand breaks at both the genomic locus and the donor vector, thereby promoting the alignment between cleavage genomic site and linearized donor using microhomology  into the genomethe cell . One sgRhomology .39. In this case, CRISPR/Cas9 is designed to cleave both the targeted genomic locus and transgene donor vector that contains long homology arms (600–900 bp each homology arm) 27. Targeted transgene integration mediated by HMEJ would not alter global gene expression profiles39. Moreover, targeted integration of a large gene payload is useful for the creation of reporter cells, production of recombinant pharmaceutical proteins, and efficient biallelic gene disruption. However, similar to MMEJ-based knock-in approaches, HMEJ requires a PAM sequence close to the CRISPR/Cas9 cleavage site in the genome.The MMEJ-based strategy was subsequently devised to an HMEJ approach by enabling more efficient targeted transgene integration using longer and more stable homology armsogy arm) . This HM2 phase in dividing cells133. Importantly, NHEJ is a predominant DNA repair pathway in non-dividing cells and post-mitotic tissues . Therefore, the efficiency of insertional mutagenesis and gene correction is limited by NHEJ that competes with HDR. In fact, NHEJ-based knock-in was recently shown to have a higher efficiency than HDR-mediated gene targeting135. In this case, double-strand DNA breaks are introduced to both the genome and donor template for mediating transgene insertion via the NHEJ repair pathway (135. The donor plasmid is linearized using Cas9 to cleave one sgRNA target site (for the single-cut donor) or two sgRNA target sites at both sides of the transgene (for the double-cut donor). However, double-cut donor is less efficient than the single-cut donor because the former will generate two DNA fragments that compete for genomic integration134. The linearized donor plasmid then is directly ligated to the broken genomic DNA ends upon NHEJ repair. NHEJ efficiently re-ligates DNA ends without mistakes and it does not require regions of homology for precise transgene insertion. However, the donor can be inserted in either orientation of the broken genomic DNA ends upon NHEJ repair. Off-target DNA double-strand breaks would also lead to random donor insertion to the unintended genomic sites. To circumvent this issue, a short homology DNA sequence bearing the Cas9 target sequence (bait sequence) is introduced onto a donor plasmid  or integrate a very large transgene  into the genome, two additional short ssODNs (~80 bp) are introduced together with self-cleaving plasmid donor and CRISPR/Cas9 targeting the genomic locus into the cell 119. Upon137. Gene splicing is manipulated by mutating specific DNA bases within splice acceptor sites or the addition of splice acceptor and donor sites to the donor DNA. For example, to add a tag to the endogenous mRNA, a donor plasmid with a tag flanked by splice acceptor and donor sites is used , and a self-linearizing repair template was recently used for gene replacement  ability43. BER accurately repairs base lesions and single-strand breaks throughout the cell cycle148. Base editor is favorable to Cas9 because it enables clean mutational knock-in by direct conversion of nucleotides in genomic DNA without inducing double-strand DNA breaks that can generate unwanted indel mutations. Nevertheless, designing multiple guide sequences of base editor to target several genomic sites in trans may still be able to lead to double-strand DNA breaks and result in the deletion of the intervening segment between two sgRNA target sites. Importantly, base editing is particularly useful for manipulating point mutations and correcting pathogenic variants in postmitotic cells and most of the somatic tissue in vivo152. Nucleotide substitutions with base editor generated much excitation partly because the HDR event is highly inefficient in non-dividing cells and the NHEJ-based editing can lead to on-target indel formation. The use of base editors also can simplify the delivery of editing machinery because no homologous DNA repair template is required to edit the genomic DNA sequence.Because the imprecise repairing of Cas9-induced DNA double-stranded breaks can lead to on-target mutagenesis and chromosomal rearrangements42. Meanwhile, adenine base editor  mediates the direct conversion of adenine to inosine, which eventually leads to an A-to-G (or T-to-C) substitution , and a rat APOBEC1 cytidine deaminase variant (R33A)  mediates the direct conversion of cytidine to uridine, which eventually leads to a C-to-T (or G-to-A) substitution 42. Meanwtitution 43. Concuvariants . In addit (R33A) . The spe156 . To broaden the targeting scope, the editing window of base editors was recently expanded from 4 or 5 nucleotides to 8 or 9 nucleotides by circularly permuting the Cas9n domain of base editors158. To narrow the editing window for single-nucleotide replacement at a specific position, high-precision base editors were engineered by optimizing the length of the linker between the deaminase domain and the Cas domain of the editor160.Base editors have also successfully been used to program exon skipping156  and rest156  are present in the editing window. Therefore, several next-generation base editors were recently engineered to address these issues. These next-generation base editors introduce desired point mutations at a target locus at significantly higher efficiency and fewer undesired by-products than a CRISPR/Cas9-mediated HDR approach43. The genome-targeting scope of these next-generation base editors is also broader than the dCas9- or Cas9n-derived base editor164.Although the base editors enable direct conversion of C-to-G base transversions and all four transition mutations  at target loci in living cells, base editing encounters several limitations. The application of base editing is restricted to point mutation substitutions. Base editing also has a limited targeting scope, low editing efficiency, and high off-target activities42. Uracil glycosylase inhibitor (UGI) blocks uracil DNA glycosylase from reversing the U:G pair to a C:G pair, thereby improving base-editing efficiency. Replacing dCas9 with Cas9n improves base-editing efficiency by inducing mismatch repair pathway to resolve the U:G mismatch into desired U:A and T:A products. To stimulate mismatch repair, Cas9n is used to nick the non-edited strand containing a G opposite the edited U. Later, APOBEC1-dCas9-UGI and APOBEC1-Cas9n-UGI were further evolved into BE4max or AncBE4max by improving nuclear localization and optimizing codon usage for maximum expression of base editors44. The BE4max and AncBE4max provide higher base-editing efficiency than the older cytidine base editors. Nevertheless, the editing window remains similar to the previously established cytidine base editors. With the phage-assisted continuous evolution of the base-editor method, these cytosine base editors were subsequently evolved into evoAPOBEC1-BE4max, evoFERNY-BE4max, and evoCDA1-BE4max165. Compared with BE4max and AncBE4max, these three evolved base editors are more efficient at editing cytosine in the GC context. EvoFERNY is also smaller than APOBEC1, thereby enabling the packaging of EvoFERNY-BE4max into the AAV vector for in vivo applications. EvoCDA1-BE4max is useful for a difficult-to-edit site or cell type. It should be noted that their editing windows and targeting scope are different from the original BE4max. Human APOBEC3G-Cas9n fusion was another recently engineered cytidine base editor to reduce unwanted bystander activities and improve precision of base editing in the CC context (repeated Cs)166. To minimize unwanted RNA-editing activity without sacrificing on-target DNA-editing activity, Cas9n-UGI was fused to non-APOBEC1 cytidine deaminases, such as an enhanced human APOBEC3A (eA3A) and human activation-induced cytidine deaminase (hAID)167. These base-editor variants also did not self-edit their own transcripts, thereby avoiding heterogeneity in base-editor coding sequences.Next-generation cytidine base editors enhance desired base editing and minimized on-target indel formation by fusing uracil glycosylase inhibitor to the base editor  and employing a Cas9n  targeting the non-edited strand43. ABE7.10 was derived from TadA*6-dCas9 variants that harbor W23L/R, P48A, and R152H/P mutations after seven rounds of evolution and engineering. These mutations abrogate base-specific enzyme–DNA interactions to broaden target sequence compatibility, thereby enhancing ABE7.10-mediated base-editing efficiency at targets that contain multiple A residues. Subsequently, ABE7.10 editor was evolved into ABEmax for more efficient base editing by improving nuclear localization, maximizing protein expression, and ancestral reconstruction of the deaminase component44. To reduce off-target RNA-editing activity, miniABEmax was engineered by removing the wild-type TadA domain and altering amino acid residues (K20A/R21A or V82G) within the mutant TadA domain of ABEmax to reduce the RNA-recognition capability167. Compared with ABEmax, miniABEmax has lower Cas9-independent RNA-editing activity but maintains Cas9-assisted on-target DNA-editing function. Notably, the self-editing activity of miniABEmax was significantly lower than ABEmax. Self-editing by base editors can lead to the generation of truncated Cas9n with intact deaminase activity, thereby inducing off-target RNA editing but not on-target DNA editing.Similar to cytidine base editor, next-generation adenine base editor  was engineered to mediate the conversion of A/T to G/C in genomic DNA169. Despite its broadened PAM compatibility, xCas9 retains high DNA specificity with minimal off-target activity. Subsequently, the base-editing efficiency of the xCas9-derived base editors  was improved by fusing the BPNLS or Gam (or both) to the N terminus of xCas9-derived base editors 45. Biparticle nucleus localization signal (BPNLS) enables efficient localization of base editors to the nucleus, while the Gam protein of bacteriophage Mu minimizes the production of undesired by-products upon base editing. More recently, NG-BE4max and NG-ABEmax base editors were generated by fusing a newly engineered SpCas9-NG variant to BE4max or ABEmax base editor170. SpCas9-NG variant recognizes minimal NG PAMs more efficiently than the xCas9 variant, thereby enabling efficient base editing with expanded targeting scope. The targeting scope and editing window of base editors have also been expanded using circularly permuted and PAM-modified Cas9n variants such as CP-CBEmax and CP-ABEmax158. In 2020, SpRY, a near-PAMless SpCas9 variant, was successfully engineered to target almost all PAMs (NRN>NYN PAMs), thereby eliminating PAM recognition constrains171. To overcome potential undesirable off-target effects due to the relaxation of the PAM requirement, a high-fidelity SpRY variant (SpRY-HF1) with improved DNA targeting specificity was subsequently engineered. Compared with dCas9, xCas9, and SpCas9-NG, SpRY-derived base editors enabled robust editing of the vast majority of disease-relevant genetic variants in the genome.The recent development of xCas9-derived base editors that recognize a broad range of PAM sequences has expanded the targeting scope in the genome172. In this case, an inducible active Cas9 with the same sgRNA as the base editor is employed. The cells with non-edited target bases remain vulnerable to Cas9 cleavage. Cas9-mediated double-strand breaks lead to the death of non-edited cells in prokaryotic cells. However, the NHEJ is the major outcome instead of cell death in mammalian cells. To enrich base-edited cells in eukaryote cells, several alternative reporter systems have been developed174. For example, a transient reporter that converts blue fluorescent protein (BFP) to GFP upon a base substitution was employed173. The GFP-positive cells can be subsequently sorted for enrichment of base-edited cells. Compared with reporters of transfection that reflect only transfection efficiency, this BFP-to-GFP conversion reporter provides more accurate information about base-editing efficiency. In another reporter system, an inactivated eGFP reporter was placed downstream of wild-type mCherry and a T2A site174. The eGFP fluorescence is restored by correcting a point mutation in eGFP transgene via base editing. In this way, mCherry allows the identification of successfully transfected cells, while eGFP enables the quantification of single base-editing efficiency. The eGFP- and mCherry-positive cells then can be sorted. These two fluorescent-based reporter systems also allow high-throughput evaluation and identification of small molecules and factors that influence base-editing efficiency. To generalize the applications of the reporter to different cell types and systems, a functional reporter system (GO system) was subsequently developed for enrichment of base-edited cells175. GO works by initiating protein expression and affecting protein translation of different reporter proteins through correction of a mutated start codon immediately downstream of a kozak sequence using a base editor. This flexible GO system has helped to expand the base-editing reporter toolbox because it can be used to induce the translation of an array of different reporters, including fluorescent proteins, antibiotic resistance, and luciferase.To enrich base-edited cells and increase the base-editing efficiency, a double-check base-editing approach is employed to exert a selective pressure against non-edited cells 172. In t46. Prime editing has lower off-target editing than Cas9 nuclease and displays higher editing efficiency with fewer by-products than the CRISPR/Cas9-mediated homologous recombination approach. Compared with homology-dependent approaches that introduce edits within 10 bp from the Cas9 cleavage site, prime editor can install point mutations at distances of more than 30 bp from the Cas9n nicked site176. Therefore, prime editor offers greater targeting flexibility than the homology-dependent approaches. Most importantly, prime editing enables correction of all types of substitutions such as transitions and transversions as well as small insertions and deletions without requiring double-strand breaks or exogenous donor DNA repair templates178. Prime editing could also complement base editing in the case of unwanted bystander edits from the presence of multiple cytidine or adenine bases within the editing window of base editors176. Furthermore, prime editing outperformed the base editor for bases positioned outside the center of the base-editing window and could be an alternative tool when the desired genomic DNA site is not targetable by the base editor.In 2019, a prime-editing strategy was developed to overcome the limitations of homology-directed gene correction and base editing46. The efficiency of prime editing is determined largely by the design of the pegRNA such as the length of primer binding site and RT template as well as the GC content, primary sequence motifs, and secondary structures of the pegRNA176. Manipulation of DNA repair machinery to favor the replacement of the non-edited strand in the DNA heteroduplex could also improve the desired editing outcome.Prime editing uses a fusion protein consisting of Cas9n and an engineered RT. Importantly, a prime-editing guide RNA (pegRNA) that both specifies the target site of sgRNA and encodes the desired edit of the RT template is used to form complexes with Cas9n–RT. As a general principle, Cas9n–RT complexed with the pegRNA first binds and nicks the target DNA . The res179. The application of primer editor, similar to that of base editors, is limited to correcting point mutations or small genetic alterations. HDR- and NHEJ-based approaches are preferred to confer large DNA insertions or deletions. The addition of the desired edit of the RT template to the sgRNA may destabilize the extended sgRNA through the activity of intracellular RNA-degrading enzymes180. Therefore, it remains impractical to add a very long RT template to the sgRNA for large genetic alterations. Moreover, the RT template in pegRNA can be incorporated in the genome during reverse transcription. It is also a challenge to co-package Cas9n-RT and its pegRNA into a single AAV vector for prime editing in vivo because of large sizes of Cas9n-RT and the lengthened sgRNA. This AAV packaging issue can be addressed by using smaller Cas9n and RT enzymes.Since prime editing and base editing do not employ any selection marker, their precise, scarless genome-editing events can be identified through the recently developed dinucleotide signature capture (DTECT) detection methodin situ, engineering antibodies, mapping protein–drug interactions, and cell-lineage tracking.CRISPR technology has recently been harnessed for modifying multiple DNA bases simultaneously and enabling genetic diversification in living cells through targeted mutagenesis in the genome. Genetic diversification is useful for establishing complex libraries of genetic variants, enabling high-throughput screening of functional variants, evolving protein structure and function 182. When dCas9-AIDx is used together with multiple sgRNAs, it expands the editing window and generates more mutations in the protospacers. The dCas9-AIDx is created by fusing dCas9 to the activation-induced cytidine deaminase (AID). AID directs somatic hypermutation to a given genomic locus by deamination of cytosine to uracil. After deamination, point mutations are generated through misread of the uracil–guanine mismatch, base-excision repair of the uracil, or mismatch-repair pathway to generate transitions and transversions near the lesion182. Targeted mutagenesis efficiency is further improved by incorporating two MS2 hairpin-binding sites to the sgRNA for recruiting multiple copies of the hyperactive AID variant (AID*Δ) to each dCas9182. Besides, targeted mutagenesis specificity of dCas9-AIDx is improved by blocking uracil DNA glycosylase181. Coupled with uracil-DNA glycosylase inhibitor, dCas9-AIDx specifically converts cytidines to thymines in the protospacer. AIDx has also been fused to Cas9 nickase for optimal nucleotide conversion181. Therefore, these targeted mutagenesis strategies create a diverse repertoire of point mutations in situ, dismissing the need to use error-prone PCR to create mutations in vitro or design a large number of DNA oligos for incorporation into a genomic locus. It also introduces a large spectrum of point mutations without generating insertions and deletions observed with active Cas9. Nevertheless, Cas9n-AIDx can induce double-strand DNA breaks when designing multiple sgRNAs to target different target DNA sites in the genome181. To improve the efficiency of base editing in GC contexts and reduce bystander activity, AID-Cas9 was later evolved into the enhanced AID-Cas9 fusion (eAID-BE4max)183.In 2016, two independent research groups developed the dCas9-AIDx fusion protein for converting cytidines or guanines to the other three bases at the desired loci 181,182. 47. Compared with dCas9-AIDx-based targeted mutagenesis approaches (editing window within five nucleotides)182, Cas9n-PolI3M is capable of continuously diversifying all nucleotides within a tunable window length (up to 350 nucleotides) at user-defined loci47. Therefore, Cas9n-PolI3M generates a larger spectrum of substitution mutations than dCas9-AIDx for large-scale genetic screenings. Cas9n-PolI3M consists of a CRISPR-guided nickase (Cas9n) and a fused nick-translating DNA polymerase (PolI3M)47. The targeted mutation rate of Cas9n-PolI3M was enhanced by creating three mutations  to Cas9n to promote the dissociation of Cas9n from DNA after nicking the target locus. In addition, a more processive DNA polymerase variant, PolI3M, was engineered to increase the length of the editing window and provide a higher targeted mutation rate than the wild-type DNA polymerase. As a general principle, Cas9n first nicks the target locus, resulting in a single-strand break in DNA  was developed for genetic diversification in living cellsk in DNA . Then Ca185. Cas9 activity poses the risk of unanticipated chromosomal rearrangement at the targeted sites and permanent, off-target mutagenesis to DNA120. In this case, RNA base-editing technology offers a safer strategy by directly editing the pathogenic transcripts without the loss of genomic information. Because RNA editing is transient, it ensures that no by-products or unwanted mutations are generated in genomic DNA that can be passed on to daughter cells during mitosis. It also useful for curing diseases without a genetic origin and mimicking genetic variants that provide a health advantage186. Because dCas13 has no targeting sequence constraints and no motif preference surrounding the target base, RNA base-editing technology helps to circumvent the issue associated with the limited targeting sites of DNA base editors. RNA base-editing technology does not rely on endogenous DNA repair pathways; therefore, it mediates efficient RNA editing even in post-mitotic cells.Pathogenic point mutations can be directly corrected in RNA transcripts instead of mutagenesis of the genomic DNA49. dCas13 is capable of binding mRNA in a nucleic acid–programmed manner, while ADAR2 (adenosine deaminase acting on RNA type 2) deaminates adenosines mispaired with cytidine bases in RNA duplexes without any protein co-factors. The deaminase domain of ADAR2-containing hyperactivating mutations (ADARDD) was later fused to dCas13b to enhance RNA-editing efficiency. The editing specificity of dCas13b-ADAR2DD was improved by structure-guided protein engineering of ADAR2DD (E488Q/T375G) to destabilize its binding to off-target RNA. Owing to the exogenous nature of dCas13b-ADAR2DD fusion proteins, host immune responses may compromise the efficacy of the RNA editing and cause side effects in vivo. The dCas13b-ADAR2DD fusion protein is also too large for packaging into the limited payload capacity of AAV vector. About two years later, dCas13b-ADAR2DD was refined to improve RNA-editing specificity and overcome immunogenicity issues associated with ectopic overexpression of ADAR by recruiting endogenous ADAR using short engineered ADAR-recruiting RNAs (arRNAs)48. The arRNA is a long-guide RNA (≥71 nucleotides) that could anneal with the target transcript to form dsRNA substrate. The dsRNA substrate then recruits endogenous ADAR proteins for targeted editing. Placing the A-C mismatch in the middle region of arRNAs results in the highest RNA-editing efficiency, while the addition of multiple A-G mismatches to each arRNA minimizes off-target editing. To further enhance RNA-editing efficiency, arRNA is fused with an ADAR-recruiting scaffold to recruit multiple copies of ADAR protein to target RNAs. A similar arRNA scaffold can also be used to recruit endogenous ADAR proteins to edit the genetic materials of RNA virus such as coronavirus in order to restrict viral propagation in the host. In fact, there was evidence for host-dependent RNA editing in the transcriptome of coronaviruses from coronavirus-infected patients187.dCas13-ADAR2 has been employed to direct adenosine-to-inosine deaminase activity to endogenous RNA transcripts 49. dCas150. ADAR2dd then was fused to dCas13 for targeted RNA editing  into a cytidine deaminase editing . The spe188. Although dCasRx alone was sufficient to promote exon exclusion by blocking access of splicing machinery189, dCasRx requires splicing regulatory domains to induce exon inclusion. Alternative slicing can be programmed using RBFOX1N-dCasRx-C or RBM38-dCasRx, a fusion of dCasRx and splicing factors (RBFOX1 or RBM38)188. The RNA-binding domain of splicing factors was substituted by dCasRx to derive the CASFx. For example, RBFOX1N-dCasRx-C or RBM38-dCasRx induces exon inclusion when bound downstream of the target exon  were developed for manipulation of gene splicingget exon . In cont186. Moreover, endogenous ADAR proteins are usually present in small amounts in most tissues for arRNA-based recruitment, thereby undermining RNA-editing efficiency in complex tissue186. The efficiency of RNA editing can be improved by using chemically modified sgRNA and a next-generation viral delivery vector or nanoparticle. For example, AAV delivery of exogenous RNA editor and guide sequence has enabled in vivo repair of mutant RNAs in mouse models of neurological disease190. Compared with gene editing, RNA editing also makes limited kinds of change to RNA. However, RNA-editing capabilities could be expanded through protein engineering to derive new RNA editors.Despite the reactiveness of RNA editing for research and therapeutic potential, RNA editing is much less efficient than gene editingVarious CRISPR-mediated homology-dependent and -independent gene knock-in and gene correction strategies have been developed. Multiplex, flexible, scarless gene insertion and replacement—together with the emerging tools for genetic diversification, prime editing, base editing, and RNA editing—can be achieved at high efficiency and specificity. We highlight structure-guided engineering of Cas9 variants that have greatly improved the gene-editing efficacy, specificity, editing window, and targeting scope. In addition, we comprehensively discuss factors involved in various DNA repair pathways enabling transgene integration and gene correction, such as Cas9 variants , fusion domains , donor type , length of homology arms , number of sgRNAs , DNA cleavage , and cleavage pattern .We discuss the choice of gene correction approaches depending on the nature of the mutations , types of mutation , copy of alleles for modification , and editing outcome (scarless modification or insertion of the selection marker to the genome for the enrichment of edited clones). We also discuss the choice of transgene knock-in approaches determined by the transgene size, donor design (the length of homology arms), plasmid donor conformation , chromatin accessibility of insertion site (open or closed chromatin), and the purpose of transgene knock-in .191. Other strategies to minimize undesired off-target mutagenesis are using light-activated, chemical-inducible, or multiple-input logic gate genetic circuits to spatiotemporal and conditional control of CRISPR/Cas9 activities192; cell type- or tissue-specific promoters and delivery vehicles , engineered high-fidelity Cas9 variants200, and rational chemical modifications to the sgRNA192; a chimeric DNA-RNA guide201; and engineered truncated sgRNA203 and sgRNA secondary structures204. Onsite mutagenesis and heterogeneity can also occur in the uncorrected population of cells following CRISPR/Cas9-induced double-stranded DNA breaks99. We also emphasize the importance of examining the potential unwanted head-to-tail insertions of donor DNA templates into the genome by HDR or NHEJ mechanisms (or both) before precisely edited alleles can be correctly identified205 and how chemical modification of both ends of the donor can help overcome this issue by enabling only one whole copy of the desired DNA to be inserted into the genome. In this fast-moving field of CRISPR-mediated gene editing, we hope to provide comprehensive information on recent developments to a broad scientific community for facilitating the application of this versatile technology.Finally, we discuss various strategies to address the potential immunogenicity associated with exogenous CRISPR fusion proteins. These include strategies to recruit endogenous catalytic domains  for gene knock-in and gene correction and controlled timing of CRISPR/Cas9 delivery and expression to minimize the off-target effects, toxicity, and immunogenicity associated with CRISPR components. Immunogenicity issue can also be circumvented by transient modulation of the genes involved in immunity by using a CRISPR-based synthetic repressor


Characterizing the humoral immune response to SARS-CoV-2 and developing accurate serologic assays are needed for diagnostic purposes and estimating population-level seroprevalence.We measured the kinetics of early antibody responses to the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in a cohort of 259 symptomatic North American patients infected with SARS-CoV-2 (up to 75 days after symptom onset) compared to antibody levels in 1548 individuals whose blood samples were obtained prior to the pandemic.Between 14–28 days from onset of symptoms, IgG, IgA, or IgM antibody responses to RBD were all accurate in identifying recently infected individuals, with 100% specificity and a sensitivity of 97%, 91%, and 81% respectively. Although the estimated median time to becoming seropositive was similar across isotypes, IgA and IgM antibodies against RBD were short-lived with most individuals estimated to become seronegative again by 51 and 47 days after symptom onset, respectively. IgG antibodies against RBD lasted longer and persisted through 75 days post-symptoms. IgG antibodies to SARS-CoV-2 RBD were highly correlated with neutralizing antibodies targeting the S protein. No cross-reactivity of the SARS-CoV-2 RBD-targeted antibodies was observed with several known circulating coronaviruses, HKU1, OC 229 E, OC43, and NL63.Among symptomatic SARS-CoV-2 cases, RBD-targeted antibodies can be indicative of previous and recent infection. IgG antibodies are correlated with neutralizing antibodies and are possibly a correlate of protective immunity. On March 11th, 2020 the World Health Organization (WHO) declared COVID-19 a pandemic. As of July 13th, 2020, the disease has caused 12,750,275 confirmed cases and 566,355 deaths globally2.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has spread rapidly around the world since first identified in Wuhan, China, in December 20194, including the magnitude and duration of responses, cross-reactivity with other coronaviruses and viral respiratory pathogens, and correlates of protective immunity following infection. A detailed characterization of antibody responses is needed to determine whether antibody-based tests can augment viral detection-based assays in the diagnosis of active or recent infections and to inform the design and interpretation of seroepidemiologic studies.Currently, our understanding of antibody responses following infection with SARS-CoV-2 is limitedIn this study, we characterize the kinetics and antibody isotype profile to the receptor binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in a longitudinal cohort of North American patients infected with SARS-CoV-2 and in pre-pandemic controls. We evaluated the sensitivity and specificity of anti-RBD responses in detecting recent infection and estimated the time it takes for cases to become seropositive (seroconversion) or return to seronegative (seroreversion). We also examined how well these responses correlated with neutralizing antibody activity directed at the S protein. Additionally, we evaluated the cross-reactivity of these responses with other coronavirus RBDs and characterize assay performance using dried blood spots.Clinical samples were obtained from individuals with PCR confirmed SARS-COV-2 infection presenting to the Massachusetts General Hospital (MGH) in Boston, MA with fever and/or viral respiratory symptoms from March to April 2020 and who met criteria for RT-PCR testing. These criteria changed over time, but included patients with severe symptoms requiring hospital admission, who had other risk factors for disease progression , or who worked or lived in a setting where infection control requirements dictated a need for testing. Additional serum/plasma samples collected September 2015 to December 2019, prior to the SARS-COV-2 outbreak, were used as controls. This included healthy adults seen at the MGH Immunization and Travel Clinic prior to travel, patients undergoing routine serology, and patients presenting with other known febrile illnesses. Plasma samples, except for the routine serology samples, were heat-inactivated at 56°C for one hour prior to analysis. DBS sample preparation is provided in the MN975262], Middle East Respiratory Syndrome (MERS) virus [GenBank: AFY13307.1], SARS-CoV-1 [GenBank: AAP13441.1], and common cold coronaviruses HKU1 [GenBank: AAT98580.1], OC229E [GenBank: AAK32191], OC43 [GenBank: AAT84362], and NL63 [GenBank: AKT07952]. Anti- RBD-specific antibody concentrations (ug/mL) were quantified using isotype-specific anti-RBD monoclonal antibodies5. A full protocol is provided in the https://www.protocols.io/view/sars-cov-2-rbd-elisa-bikbkcsn).The ELISA assays measured IgG, IgA, and IgM responses to the receptor binding domain of the spike protein (RBD) from SARS-CoV-2 [GenBank: 6, which is a strong correlate of protective immunity in challenged rhesus macaques7. We expressed results from this assay as the antibody titer required to neutralize 50% of the SARS-CoV-2 pseudovirus (NT50).To determine the SARS-CoV-2 neutralization activity of our plasma samples, we used a lentivirus pseudoneutralization model as previously described8, we allocated both cases and controls among 10-folds to calculate a pooled cross-validated AUC (cvAUC). Using the isotype cut-offs defined by the maximum concentration (ug/mL) found among pre-pandemic controls , we estimated sensitivity and bootstrap 95% confidence intervals.We first explored how cutoffs of individual isotypes  performed in identifying previously infected individuals. We compared measurements from pre-pandemic controls, with those taken at any time, ≤7 days, 8–14 days,>15–28 days, and >28 days after the onset of symptoms. Using a previously described cross-validation procedureWe explored how combining multiple isotype-specific responses with a random forest classification models performed identifying previously infected individuals. We repeated the same procedures for cross-validation and assessed variable importance within these different models using a permutation test–based metric, mean decrease in accuracy.9. All time-to-event data were assumed to be Weibull distributed with bootstrapped 95% confidence intervals estimated.Using the cut-offs defined earlier, we modeled the time required to become seropositive (seroconversion) and return to becoming seronegative (seroreversion). Using individual level interval-censored data, we fit non-parametric (i.e Turnbull’s Estimator) and parametric accelerated failure time models using the icenReg R packageAll analyses were completed using R (Version 3.6.1) within Rstudio (Version 1.2.5019).We measured RBD responses in two cohorts: 1) symptomatic patients who tested positive for SARS-CoV-2 by PCR (n = 259) and 2) healthy  and febrile controls (n = 33) collected prior to the SARS-CoV-2 outbreak. The majority of SARS-CoV-2 positive cases were severe , male (62%), and older (median age: 58) . Most prMost cases eventually had evidence of elevated antibodies to SARS-CoV-2 compared to pre-pandemic controls . From daEach antibody isotype was predictive of infection, and the cross-validated AUC (cvAUC) of antibody testing for each antibody isotype increased to above 97% during the period of 15–28 days after symptom onset . The cvAThe discriminatory power of IgG alone was high after 14 days; however, we found the accuracy of ELISA-based identification of recent infections could be improved by adding IgM and/or IgA at the earlier phases of infection . The ranOf 94 cases with samples after 20 days post-symptoms, most had evidence of seroconversion for all isotypes . The estimated median time to seroconversion from symptom onset was comparable across antibody isotype: 10.9 days (95% CI:10.0–11.9) for IgG, 11.8 days (10.7–13.1) for IgA and 12.1 (10.8–13.7 days) for IgM . On averWe measured pseudoneutralizing antibodies targeting the SARS-CoV2 S protein in 88 samples from 15 individuals collected between 0 and 75 days post-symptoms . Over thWe evaluated antibody responses to RBDs derived from spike proteins of endemic human coronaviruses (CoVs) , severe acute respiratory syndrome coronavirus (SARS-CoV-1) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) . We did Since this assay could be used in large serosurveys, we also evaluated the assay with dried blood spot eluates in a subset of patients  and pre-pandemic controls (n=20). The anti-RBD IgG DBS measurements had a high degree of linear correlation in both cases and control plasma  and sensitive (97%) marker of infection after 14 days from onset of illness. IgG seropositivity was sustained in patients up to 75 days (last time point measured), and the concentration of these antibodies was highly correlated with pseudovirus NAb titers. In contrast, IgM and IgA responses to RBD were short-lived and with many individuals seroreverting within two months of the onset of illness. Longer term follow-up is needed to determine the duration of IgG responses.10, and our results suggest that the detection of antibodies against the SARSCoV-2 RBD by ELISA is not likely to contribute significantly to the early diagnosis of COVID-19. Even models incorporating all isotypes had limited predictive capacity during the first week of illness. However, our results do demonstrate that the detection of IgM and IgA responses improved the sensitivity of serologic testing within two weeks of the onset of symptoms. Beyond two weeks after symptom onset, supplementing viral detection assays with antibody-based testing methods clearly increases sensitivity in diagnosing recent infection12, particularly as the sensitivity of RT-PCR for SARS-CoV-2 infection wanes13. Our results, also, show that the early seroreversion of IgA and IgM responses could be useful in the future in distinguishing previous infection from acute COVID-19. Taken together, these findings suggest specific and clearly defined applications for serologic testing of RBD responses in the clinical setting.RT-PCR based detection of SARS-CoV-2 is sensitive early in the first week after the onset of symptoms15. In contrast, our study, based on a large sample of cases and controls should provide significant confidence in the potential contribution of serologic measures in public health efforts to identify transmission hot-spots and at-risk populations. In addition, the lack of cross-reactivity of SARS-CoV-2 RBD with common cold coronaviruses should provide additional confidence in the specificity of the assay. Longer term characterization of IgG responses, however, is still needed to assist in design and interpretation of serosurveys.Testing for anti-SARS-CoV-2 RBD antibodies can also be applied in seroepidemiologic studies, even in areas of low prevalence, given their excellent specificity and defined kinetics. Variation in the performance of commercial serologic tests and confusion about the role of antibodies as biomarkers of past infection versus protective immunity has led to widespread misperception that antibody testing may not be accurate or useful13, which may lead to false negatives if our proposed assay thresholds are used. Individuals with mild or asymptomatic infection may also serorevert quicker than symptomatic individuals13. The gradation of responses by disease severity has been found in other infections, including SARS-CoV-213 and MERS-CoV infection16. An association between disease severity and the kinetics of the antibody response is also suggested by our finding that individuals with more severe disease, who required ICU-level care, seroconverted earlier than individuals who did not require ICU-level support.One notable limitation of our study was that our cohort of individuals with SARS-CoV-2 infection was skewed toward adults with clinically significant disease or with risk factors for disease progression. Individuals with mild or asymptomatic infection have been shown to develop less robust antibody responses17. In addition, in vaccinated rhesus macaques challenged with SARS-CoV-2 infection, neutralizing antibodies directed at the S protein were also a strong correlate of protective immunity6. Thus, neutralization titers, in the absence of other known markers, has become a de facto immunologic marker of protection pending further investigation; however, whether there is a threshold titer that is robustly associated with protection remains unclear. Nonetheless, it is notable that anti-RBD IgG antibodies were strongly correlated with the neutralizing antibodies associated with protection in vaccinated macaques6. This correlation with neutralizing titers was stronger than observed for other previously tested commercial serologic assays18.It is important to note that our assay identifies individuals with recent SARS-CoV-2 infection but does not provide information about whether individuals are protected from subsequent infection. Optimal immunologic correlates of protection for SARS-CoV-2 remain unknown in humans. In many human challenge studies of common cold coronavirus infection, the presence of pre-existing neutralizing antibodies has been associated with protection against the development of symptomatic infection and with decreased viral shedding19 and has the potential to facilitate accurate diagnosis in clinical settings and the implementation of population-based studies of previous infection globally. While the association between RBD-IgG with neutralizing titers and the persistence of these antibodies at late time points is encouraging, further work is needed to define the optimal antibody mediated correlates of protective immunity.Our results, therefore, provide strong support for the application of anti-RBD antibodies as a marker of recent SARS-CoV-2 infection. This approach meets the CDC’s interim guidelines for serologic testing1


In conclusion, we found that the expression of miRNAs depends on the age of the patient and/or the severity of the disease, suggesting that miRNAs may contribute to the regulation of inflammation in UC and could be useful biomarkers in the surveillance of disease severity.Despite significant evidence that the expression of several microRNAs (miRNAs) impacts disease activity in patients with ulcerative colitis (UC), it remains unknown if the more severe disease phenotype seen in pediatric onset UC can be explained by an altered miRNA expression. In this study, we assessed the relationship between miRNA expression, age, and disease severity in pediatric and adult patients with UC. Using RT-qPCR, we analyzed the expression of miR-21, miR-31, miR-126, miR-142 and miR-155 in paraffin embedded rectum biopsies from 30 pediatric and 30 adult-onset UC patients. We found that lesions from adult patients had significantly higher expression levels of miR-21 compared to pediatric patients and that the expression levels of miR-31  and miR-155 (pediatric patients only) correlated inversely with histological assessed disease severity. Using  Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by a chronic inflammation of the colon. The extent of the disease varies, but adult-onset UC (aUC) typically presents with a distal proctitis (limited disease extent). By contrast pediatric onset UC (pUC) typically presents with the entire colon affected (extensive disease extent) . From thSince the etiology of UC is poorly understood, it is unknown whether the difference in phenotype between pUC and aUC is caused by a fundamental difference in disease pathogenesis. It is thought that UC develops due to an aberrant immune response towards the intestinal microbiota in genetically disposed individuals . InteresIn aUC, the expression of several miRNAs is altered in colon mucosa –19 and b. reported a panel of six miRNAs (including miR-21) to differentiate CD from UC [in situ hybridization (qISH), Thorlacius-Ussing et al. [MiRNA-21 is likely the most extensively described miRNA in UC . MiR-21  from UC , and usig et al.  found siSeveral studies have reported on the role of other miRNAs in IBD in general, both exploring mechanistic roles and the potential role of miRNA as biomarkers and drug targets , 6, 24. Based on the fact that the existing literature consistently reports differential miRNA expression in UC patients compared to healthy controls , 14, 31,de novo diagnosed pediatric patients (new pUC), previously diagnosed pediatric patients (known pUC), de-novo diagnosed adult patients (new aUC), and previously diagnosed adult patients (known aUC).We included 60 patients equally distributed among the following four groups: Baseline characteristics are presented in The expression levels of miR-21 in pUC patients were significantly lower than in aUC patients . No difference was found between patients in histological assessed remission compared to those with active disease for both pUC and aUC study groups. Among the aUC, males had higher expression levels than females . No correlation was found between miR-21 measured by RT-qPCR and Geboes score .By ISH, we found a focal upregulation of miR-21 in association with inflamed crypts with similar expression patterns in all study groups .de novo and previously diagnosed patients, the correlation between miR-21 expression and Geboes score was higher in previously diagnosed patients . There was no significant correlation between the miR-21 qISH expression level estimates and the RT-qPCR based expression levels  with patients with histological activity, we found that pUC patients in remission had increased expression levels compared to pUC patients with active disease . MiR-31 negatively correlated with Geboes score . When stratifying by time of diagnosis, miR-31 correlated with Geboes score in previously diagnosed patients .When comparing expression levels in pUC with aUC, and the patients with active disease with remission, we found no differences in the expression levels by RT-qPCR analysis.By ISH, miR-126 was associated with the vascular endothelial cells  with simde novo and previously diagnosed patients, the correlation increased in previously diagnosed patients . The qISH expression estimates were decreased in patients in histological remission compared to patients with active disease . The miR-126 expression estimates did not correlate with the RT-qPCR results  , p=0.002There was no difference in the miR-142 expression levels comparing pUC vs aUC. PUC in histological remission had significantly increased expression levels compared to pUC with histological activity . In previously diagnosed UC patients, miR-142 levels correlated negatively with Geboes score .de novo diagnosed and previously diagnosed, miR-155 expression levels negatively correlated with the Geboes score in previous diagnosed patients only .There was no difference in the miR-155 expression levels comparing pUC vs aUC. The expression levels of miR-155 were significantly increased in pUC in histological remission . In pUC, miR-155 negatively correlated with Geboes score . When stratifying all patients by For unknown reasons, pUC presents with a more extensive disease than aUC and requires a more aggressive treatment regimen to induce and maintain remission –3. Becaund line therapies [. [Our main objective was to assess the differences in miRNA expression levels in rectal biopsies between pUC and aUC patients. Using RT-qPCR, we found that miR-21 was increased three-fold in aUC. miR-21 has repeatedly been reported to be increased in inflamed tissue in both pUC , 38, 39 herapies . Møller apies [.  studied Interestingly, DSS-induced colitis in miR-21 deficient mice was decreased compared to that in wild type mice and had reduced levels of TNF- α [. [It is noteworthy that the miR-21 expression estimates obtained by RT-qPCR and qISH did not correlate, and neither were the significant correlation of miR-21 with disease parameters replicated with the two methods. In the IBD study by Thorlacius-Ussing et al. , miR-21 . , whereas. . We therOur data also showed that the miR-21 levels measured by RT-qPCR were higher in males compared to females. A similar sex-related difference was not found among the pediatric patients, however, similar sex-related miR-21 differences have been reported in CRC and hypertrophic heart disease –49. Our QISH is rarely used to assess miRNA expression in UC, but the technique has a clear advantage over RT-qPCR including a visual inspection of the expression in the tissue and cellular compartments . miRNA eMiR-142 was found to be negatively correlated with histologically assessed severity of disease in previously diagnosed patients, and therefore seems to be associated with an anti-inflammatory response in patients with longer duration of disease. The association with disease activity could potentially be explained by reduced levels of miR-142 in T-cells upon activation . miR-142., miR-31 and miR-155 were both shown to target the IL-13 receptor α-1 mRNA [Both miR-31 and miR-155 were negatively correlated with the histological assessment of the disease and the strongest correlations were found in pUC (miR-155) and in previously diagnosed patients (miR-31), when stratifying according to patient characteristics. Moreover, the expression levels were significantly increased in patients in histologically assessed remission. In a study by Gwiggner et alα-1 mRNA , which iα-1 mRNA . When coα-1 mRNA  reportinα-1 mRNA , 60.The main strength of this study is the prospective inclusion of well-characterized patients of which half were newly diagnosed and therefore included before treatment was initiated. This allowed us to correlate the miRNA expression levels with clinical features, which is often lacking in the existing miRNA literature, and make the results from the present study more easily applicable to the problems faced in the daily clinical work with IBD patients. In conclusion, in prospectively collected FFPE rectal biopsies we found different expression levels and patterns of the examined miRNAs in pUC and aUC, which indicates differences in the etiology of pUC and aUC that might help explain the more severe disease course reported in pUC. The possible involvement of miRNA in the regulation of inflammation may compromise the efficacy of IBD therapies and calls for combined therapies directed towards partly overlapping pathways. Further studies on miRNA expression may help identify such novel relevant pathways and hopefully targets.All patients gave written consent before inclusion. If the patient was < 18 years old at time of inclusion, consent was given by both parents / guardians. The investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guideline and has been approved by the National Ethical Committee (jr. nr.: H-16049534).In this prospective study, we obtained rectal biopsies from pediatric and adult patients scheduled for endoscopy (colonoscopy or sigmoidoscopy) due to either suspected UC or a flair of a previously diagnosed UC (diagnosed >one year prior to the present endoscopy). The biopsies were obtained from patients hospitalized from July 2017 to April 2018. Exclusion criteria were young age (< five years), previous cancer diagnosis, previous bowel resection and adult UC patients who were originally diagnosed < 18 years of age. Prior to endoscopy, patients symptoms were scored by the Pediatric Ulcerative Colitis Activity Index (PUCAI) and the Simple Clinical Colitis Activity Index (SCCAI) for pediatric and adult patients, respectively , 62. At The biopsies were formalin fixed and paraffin embedded and 3 μm sections stained with hematoxylin and eosin. The histological severity were graded as follows (the Geboes score ): 0, strRNA Purification. Three paraffin sections of 5 μm thickness from each sample were placed in a 1.5 ml Eppendorf tube. RNA purification was performed using the miRNeasy FFPE Kit (Qiagen) according to the manufacturer’s instructions. In short, sections were deparaffinized in xylene and methanol, followed by permeabilization using Proteinase-K treatment and digestion with DNase to eliminate endogenous DNA fragments. RNeasy MinElute spin columns (Qiagen) were used for total RNA purification and finally eluted using RNase free-water. The RNA yield was determined using a NanoDrop 1000 (Thermo Scientific) and varied from 4 to 30 ng/μl.For Reverse transcription quantitative polymerase chain reaction, we used MiRCURY LNA miRNA PCR assays for the determination of miRNA expression levels. Complementary DNA (cDNA) was obtained by reverse transcriptase (RT) incubation on 5 ng total RNA following the manufacturer’s instructions. For the miRCURY LNA SYBR Green PCR Kit we used primer pairs for hsa-miR-21-5p (miR-21), hsa-miR-31-5p (miR-31), hsa-miR-126-3p (miR-126), hsa-miR-142-5p (miR-142) and hsa-miR-155-5p (miR-155) (Qiagen). The RT-qPCR expression level  of hsa-miR-103a-3p (miR-103) was used as endogenous reference miRNA based on previous observations [in situ hybridization analyses, we delineated a region of interest (ROI) of the total tissue area in each slide using image analysis software  as also described previously [2. For each slide, we obtained area fractions of the blue ISH signal relative to the total tissue area, representing the expression level estimates of the quantitative image analysis of in situ hybridization (qISH). From duplicate sections we used the average of the two estimates in the statistical analyses. An overview of the image analysis process is presented in Paraffin sections were cut at 5 μm thickness and placed on Super Frost Ultra Plus slides (Menzel Gläser). Slides were deparaffinized with xylene and rehydrated through decreasing concentrations of ethanol. Slides were placed on the Ventana Discovery Ultra Instrument (Roche) for automated hybridization . In shoreviously . The ave–(∆Ct) method [Unless otherwise stated, descriptive data are presented as median and either interquartile range (IQR) or range for continuous variables and frequencies and percentages for categorical variables. RT-qPCR data was analyzed by the 2) method  using miSupplementary Figures


The ubiquitin-proteasome pathway and autophagy-lysosome pathway are two major routes for clearance of aberrant cellular components to maintain protein homeostasis and normal cellular functions. Accumulating evidence shows that these two pathways are impaired during cerebral ischemia, which contributes to ischemic-induced neuronal necrosis and apoptosis. This review aims to critically discuss current knowledge and controversies on these two pathways in response to cerebral ischemic stress. We also discuss molecular mechanisms underlying the impairments of these protein degradation pathways and how such impairments lead to neuronal damage after cerebral ischemia. Further, we review the recent advance on the understanding of the involvement of these two pathways in the pathological process during many therapeutic approaches against cerebral ischemia. Despite recent advances, the exact role and molecular mechanisms of these two pathways following cerebral ischemia are complex and not completely understood, of which better understanding will provide avenues to develop novel therapeutic strategies for ischemic stroke. Protein homeostasis, the correct balance between production and degradation of proteins, is essential for cell function, development, and survival. Protein homeostasis also plays a critical role in cerebral ischemia. Protein homeostasis dysfunction has been implicated in the development and progression of neurological impairment in cerebral ischemia. Pharmacologic activation of the ATF6 arm of the unfolded protein response (UPR) reprograms cellular protein homeostasis and confers global neuroprotection in cerebral ischemia-reperfusion injury . L-2-OxoThe ubiquitin-proteasome pathway and autophagy-lysosome pathway are two major protein degradation systems in all eukaryotic cells to degrade a broad array of misfolded proteins. The ubiquitin-proteasome pathway is extremely efficient for the degradation of short-lived proteins and misfolded soluble proteins, while the autophagy-lysosome pathway is responsible for eliminating long-lived proteins, insoluble proteins, and certain whole organelles. The two highly regulated pathways do not only maintain protein homeostasis, but also mediate necrosis and apoptosis. In this review, we will focus on recent discoveries of the ubiquitin-proteasome pathway and autophagy-lysosome pathway and the crosstalk between them in the pathogenesis and treatment of cerebral ischemia and attempt to shed new light on the design of effective therapy strategies against cerebral ischemia injury-associated diseases.The expression level of ubiquitin conjugates is greatly increased in plaques, especially unstable plaques, indicating that the ubiquitin-proteasome system is involved in the development of atherosclerotic plaques in intracranial arteries . After hAfter transient cerebral ischemia, ubiquitin-conjugated proteins accumulate in Triton-insoluble aggregates. 763 peptides to 272 proteins, including proteins involved in important neuronal functions and signaling pathways, were highly enriched in these aggregates [β1i, β2i, and β5i. The expression of the immunoproteasomal subunits, 20S β1i and β5i, is increased remarkably in the parietal cortex and hippocampus after transient cerebral ischemia, which is associated with a great increase in ubiquitinated proteins. The immunoproteasome induction after cerebral ischemia is supposed to play a critical role in coping with the damaged proteins and thus may be of great importance in neuronal survival and death [Immunoproteasome, a subtype of proteasome, contains three major catalytic subunits: nd death . The pland death .α (HIF-1α) abundance [Immunoproteasome inhibition reduces infarction volume and attenuates inflammatory reaction after cerebral ischemia in rats, suggesting that selective immunoproteasome inhibitors may be promising candidates for the treatment of ischemic stroke . In a rabundance . PR-957,bundance .After transient cerebral ischemia, the depletion of ubiquitin and the accumulation of its mutant form (ubiquitin(+1)) may inhibit the ubiquitin-proteasome pathway, which results in delayed neuronal death of CA1 pyramidal neurons directly or indirectly . MeanwhiProtein aggregation has been proven to be a pathological basis responsible for ischemia-induced neuronal death. Therefore, enhanced removal of damaged proteins is supposed to be a promising therapeutic strategy for the treatment of ischemia-caused neuronal injury. Ubqln, a ubiquitin-like protein, mediates degradation of damaged proteins. Ubqln overexpression greatly inhibited the accumulation of protein aggregates and conferred neuroprotection against ischemia-induced cerebral injury . OverexpDeubiquitinating enzymes (DUBs) are a large group of proteases that negatively regulates proteasome activity. The ubiquitin-specific protease 14 (USP14), one of the DUBs, is associated with the proteasome and regulates protein degradation. The USP14 inhibitor ameliorates neuronal injury and confers neuroprotection after cerebral ischemia-reperfusion insult in mice . UbiquitProteasome dysfunction leads protein aggregation after cerebral ischemia. Trehalose inhibits transient cerebral ischemia-induced protein aggregation via preservation of proteasome activity . PropofoMicroRNA-124 confers neuroprotection in focal cerebral ischemia through inhibiting the deubiquitinating enzyme USP14 expression and reducing the expression level of REST . Parkin Ischemic preconditioning protects against transient cerebral ischemic-induced damage through inhibiting ubiquitin aggregation . Ischemiα degradation in ischemic neurons is mediated by 20S proteasomes. Proteasomal inhibition confers neuroprotection against cerebral ischemia through HIF-1α stabilization [α accumulation [Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to hypoxia and can determine the fate of neurons during cerebral ischemia. HIF-1lization . The novmulation . Preservmulation .α through ubiquitin-proteasome system-mediated degradation [α expression, reduces neuronal autophagy, and thus protects neurons against ischemia-reperfusion-induced damage [Early 2-methoxyestradiol (2ME2) administration inhibits neuronal HIF-1radation . Dexmeded damage .Cellular prion protein protects neurons against ischemic-induced damage, promotes angioneurogenesis, and enhances neural progenitor cell homing through inhibiting proteasome activity . Ginsenoγ-secretase blocker DAPT reduces blood-brain barrier (BBB) permeability during permanent brain ischemia, which is the result from decreased ubiquitination and degradation of occludin [rAAV8-733-mediated gene transfer of CHIP/Stub-1 reduces the expression of ubiquitinated proteins, which contributes to the prevention of hippocampal neuronal death in an animal model of brain ischemia . The γ-soccludin . The nucoccludin  , a ubiquitin-like protein, mediates posttranslational protein modifications and is involved in the regulation of a myriad of stress responses. Cerebral ischemia-reperfusion increases the aggregation of SUMO, which plays an important role in the altered protein dynamics after brain ischemia . Focal cNeuron-specific knockdown of SUMO inhibits global gene expression response and exacerbates the functional outcome in a mouse model of transient forebrain ischemia . SUMOylaSUMO-specific protease 1 (SENP1) deconjugates SUMO from modified proteins and confers neuroprotection in cerebral ischemia. SENP1 inhibits SUMO1 conjugation and thus protects against ischemia-reperfusion-induced apoptosis . URB597 SUMOylation of NaV1.2 channels contributes to the early response to acute hypoxia in central neurons . IschemiHypothermia confers early neuroprotective effects after middle cerebral artery occlusion in rats, which is involved in the protein conjugation of SUMO2/3 . HypotheWhen the ubiquitin-proteasome pathway is overloaded, excessive misfolded proteins will be removed by the autophagy-lysosome pathway. Depending on the BAG1/BAG3 ratio and expression level of HDAC6, a switch from the ubiquitin-proteasome pathway to the autophagy-lysosome pathway occurs between 10 and 30 min during cerebral ischemia . The lysLysosomal Psap processing is found to be changed after cerebral ischemia from proteomic analysis of synaptosomal protein expression. Cerebral ischemia adversely affects the synapse, which may be involved in regulating the neuronal viability after ischemic stroke . The disCerebral ischemia significantly increases autophagosomes and autolysosomes in hippocampal CA1 and DG neurons. Transient cerebral ischemia-induced protein aggregation on subcellular organelle membranes could result in multiple organelle damage, accompanied by delayed neuronal death . DNA damAbsence of the circadian clock protein PER1 inhibits the hippocampal autophagic machinery and contributes to vulnerability to cerebral ischemia injury . Neonataβ-catenin pathway and thus exerts protective effect in neural ischemia and hypoxia [Autophagy plays diverse roles in cerebral ischemia-reperfusion injury . Mitopha hypoxia .The autophagic-lysosomal pathway is activated in injured astrocytes following glucose and oxygen deprivation and focal cerebral ischemia, which at least partly account for decreased survival of astrocytes . AsphyxiRIP1K activates the autophagic-lysosomal pathway and induces necroptosis, which may account for the neuronal and the astrocytic cell death after cerebral ischemia . The endIschemia-reperfusion induces autophagosome formation and inhibits autolysosome degradation. Mitofusin 2, a mitochondrial fusion protein, enhances autophagosome formation and promotes the fusion of autophagosomes and lysosomes and, thus, attenuates ischemia-reperfusion-induced damage . Rab7b, Sphingosine kinase 2 exerts a neuroprotective effect in cerebral ischemia. It could interact with Bcl-2 via its BH3 domain, thereby dissociating it from Beclin-1, and activating autophagy subsequently . PhosphoResveratrol induces Sirt1-dependent autophagy, which thereby suppresses NLRP3 inflammasome activation, and thus protects against cerebral ischemia-reperfusion injury . HyperhoAutophagosome accumulation is increased after oxygen-glucose deprivation insult, which is mediated by increased autophagosome formation and decreased autophagosome clearance. Sevoflurane postconditioning protects against oxygen-glucose deprivation injury through inhibition of autophagosome accumulation . CerebraMild hypothermia confers protection against hippocampal neuronal injury after oxygen-glucose deprivation/reperfusion, which is mediated by promoting lysosomal function and autophagic flux . Remote miR-207 and miR-352 play important roles in regulating cerebral ischemic damage and spontaneous recovery through the lysosomal pathway. miR-207 mimics could decrease the expression of cellular lysosome and autophagosome and, thus, induce the expression of autophagic vacuoles . Dapsone2- cyclopentanone (DMBC), a novel synthetic small-molecule compound, inhibits the release of cathepsin B from the lysosomes into the cytoplasm after cerebral ischemia, thereby protecting neurons against ischemic injury . CA-074MLycium barbarum polysaccharide protects against ischemia-reperfusion injury in primary cultured hippocampal neurons, which is mediated by inhibition of apoptosis and autophagic cell death through the PI3K/Akt/mTOR pathway [Fingolimod inhibits ischemia-induced neuronal autophagy via the mTOR/p70S6K signaling pathway and confers neuroprotection against cerebral ischemic insult . Lycium  pathway . Cornin  pathway  (Table 5Hyperbaric oxygen (HBO) suppresses autophagy activity and inhibits apoptosis and necrosis levels, thereby conferring neuroprotection against ischemic brain injury .Sevoflurane postconditioning suppresses the activation and release of lysosomal cathepsin B and alleviates reactive astrogliosis and glial scar formation after cerebral ischemia-reperfusion .Ubiquitylation is a common component that guides misfolded protein to the proper degradation system for both the ubiquitin-proteasome pathway and the autophagy-lysosome pathway . BesidesOn the one hand, initial evidences about functional links between the ubiquitin-proteasome pathway and the autophagy-lysosome pathway reveal that inhibition of one can result in a compensatory upregulation of the other pathway . This caOn the other hand, mitophagy is also a prominent biological phenomenon that involves both the ubiquitin-proteasome pathway and the autophagy-lysosome pathway since the ubiquitin-proteasome activity has been proven to be a prerequisite in the preparation of selective autophagy for mitochondria . This scCollectively, this review provides novel insights into the role of the ubiquitin-proteasome pathway and the autophagy-lysosome pathway after cerebral ischemia. Cerebral ischemia leads to dysfunction of these two pathways, which contributes to misfolded protein accumulation and neuronal necrosis and apoptosis ultimately. Regulation of these two pathways may be an important mechanism for the neuroprotective effects of many therapeutic agents and approaches against cerebral ischemia.The challenge in the future research is to determine the key factors that mediate degradation of apoptosis and oxidative damage-related proteins, as well as the specific receptors that mediate the degradation of mitochondria, the endoplasmic reticulum, and Golgi apparatus through the autophagy-lysosome pathway in cerebral ischemic injury. To achieve this, mass spectrometry can be employed to quantitatively analyze proteins that are altered on organelles during cerebral ischemia, especially those whose expression is increased.G76V-GFP, an ubiquitin-proteasome system reporter, for monitoring the role of ubiquitin-proteasome-dependent proteolysis [At the same time, future researches will also need to clarify the dynamic changes of the ubiquitin-proteasome pathway and the autophagy-lysosome pathway at different time points during cerebral ischemia, which will help us to determine their specific role in cerebral ischemic injury. As we know, there are transgenic mice expressing Ubteolysis , transgeteolysis  and a miteolysis , which w


Falls are the leading cause of injury in older adults; one in four older adults fall each year. The Otago Exercise Program (OEP) is an evidence-based fall prevention program that has been shown to reduce fall risk factors. However, exercise dosage is not known. The purpose of this study was to investigate the effectiveness of different frequencies of an OEP-based program on fall risk factors, fear of falling, and depression in older adults. Of 62 subjects initially recruited at community centers, 28 subjects met inclusion criteria and were assigned to control (Con), once-weekly (Grp 1), or twice-weekly (Grp 2) intervention groups based on subject attendance. Intervention consisted of a 12-week OEP-based program. Pre-intervention dependent variables included: 4-Stage Balance Test, 30-Second Chair Stand, Timed Up and Go (TUG), Geriatric Depression Scale (GDS), and Modified Fall Efficacy Scale (MFES). After 12 weeks, post-intervention testing assessed changes in these variables. Preliminary analysis of data using mixed design ANOVA (p.05) indicated significant changes between and within all groups for TUG and 30-Second Chair Stand. Results also suggested all groups improved on all variables with a direct relationship to exercise frequency. Furthermore, Grp2 improved more than Grp1 and Con in the 4-Stage Balance Test. These findings suggest an OEP-based falls prevention program performed 2x/week is more beneficial in decreasing fall risks and increasing lower extremity muscle strength than 1x/week. Moreover, results imply exercise frequency may be important in improving the magnitude of select falls risks variables.


Using data from 2,923 Chinese older immigrants in Chicago, this study aims to identify different patterns of coping repertoires of older immigrants, based on a combination of individual, family and community coping resources, and the optimal coping repertoire that is associated with the best psychological outcomes. The results of Latent Class Analysis revealed four types of coping repertoires: low-resource (43%), spouse-oriented (32%), community-oriented (15%), and multi-source coping repertoire (10%). Overall, immigrants who had multi-source coping repertoire reported the best psychological outcomes. However, the influence of coping repertoires varied based on specific adversities. Having community-oriented coping repertoire was more protective for widowed immigrants, whereas spouse- or community-oriented coping repertoire was more protective for those with poor health. For less-acculturated older immigrants, having community-oriented coping appears most beneficial to their well-being; and for older immigrants who perceived low filial support from their children, having multi-source coping was associated with better psychological well-being.


Severe coronavirus disease 2019 (COVID-19) causes a hyperactivation of immune cells, resulting in lung inflammation. Recent studies showed that COVID-19 induces the production of factors previously implicated in the reawakening of dormant breast cancer cells such as neutrophil extracellular traps (NETs). The presence of NETs and of a pro-inflammatory microenvironment may therefore promote breast cancer reactivation, increasing the risk of pulmonary metastasis. Further studies will be required to confirm the link between COVID-19 and cancer recurrence. However, an increased awareness on the potential risks for breast cancer patients with COVID-19 may lead to improved treatment strategies to prevent metastatic relapse. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is spreading in a world where cancer prevalence is rapidly growing, raising concerns about potential interactions between the two diseases. SARS-CoV-2 recruits proteins involved in cellular replication, DNA damage, metabolism, and epigenetic regulation that are also implicated in cancer pathogenesis . At the SARS-CoV-2 infection induces the death of airway epithelial cells with consequent release of damage-associated molecular patterns (DAMPs). DAMPs trigger the production of inflammatory cytokines and chemokines, thus recruiting monocytes, neutrophils and T cells to the lungs Fig. . In the Several factors involved in COVID-19 may play a role in the reawakening of dormant tumor cells. The strongest evidence points to NETs and neutrophils, which are emerging as important players in COVID-19 pathogenesis. NETs involvement in COVID-19 was first proposed upon observation of intense neutrophilic infiltration in the lungs of autopsied COVID-19 patients . The preHypoxia, which arises in the blood and tissues of COVID-19 patients upon respiratory distress and thrombosis, is a poor-prognosis microenvironmental hallmark of solid tumors. In breast cancer, hypoxia has been shown to be responsible for DCCs generation by promoting the expression of genes implicated in dormancy, drug resistance, stemness, and EMT . TherefoFinally, recent studies on the long-term clinical outcomes of COVID-19 showed a high incidence of persistent symptoms after the acute disease [Ongoing clinical studies that include an assessment of the long-term effects of COVID-19 on cancer patients  will cla


A 79-year-old man with chronic atrial fibrillation underwent single-chamber His-bundle pacemaker implantation. The post-implant electrocardiogram (ECG) demonstrated selective His-bundle capture, with a narrow paced QRS and repolarization pattern similar to that of the baseline ECG. Furthermore, repolarization changes prototypic of ventricular pacing did not occur with selective His-bundle capture. While His-bundle pacing, with or without selective His-bundle capture, can preserve physiologic patterns of depolarization, only His-bundle selective pacing can preserve intrinsic ST- and T-wave patterns. Thus, the maintenance of physiologic repolarization may have various advantages, including accurate interpretation of ECG changes that are not generally interpretable in the setting of ventricular pacing. Owing to the anticipation of a high burden of ventricular pacing, His-bundle pacing was considered; the patient was subsequently referred for single-chamber pacemaker implantation.A 79-year-old man with chronic atrial fibrillation (AF) presented with progressive exertional dyspnea. On physical examination, he was found to be bradycardic, and demonstrated signs of mild congestive heart failure. Outpatient Holter monitoring revealed the presence of persistent AF, with slow ventricular rates and pauses up to 7.4 s. Additionally, an echocardiogram demonstrated normal biventricular function, with severe left atrial enlargement, and a 12-lead electrocardiogram (ECG) revealed atrial tachycardia with a slow ventricular rate and a narrow QRS with chronic ST abnormalities A single-chamber His-bundle pacemaker was implanted using a SelectSecure™ Lead Model 3830 , delivered via a C315HIS fixed shape sheath catheter . During lead implantation, the lead was fixated after a His-bundle electrogram was observed on the ventricular sensing electrode, and following pacing’s production of a narrow QRS morphology and latency period from pacing to QRS complex.. At higher pacing outputs, non-selective His-bundle pacing occurred, together with a pseudo-delta wave indicating fusion between His-bundle and local myocardial capture. Secondary ST-depression and T-wave abnormalities were also present . Notably, the repolarization changes that can be typically seen with ventricular pacing were not present when selective His-bundle was achieved. During early clinical follow-up, a high degree of ventricular pacing with selective His-bundle capture was noted, and the patient reported improvement in both his symptoms and functional capacity.The patient’s post-implant ECG demonstrates ventricular pacing, with a paced QRS and repolarization pattern similar to that of the baseline ECG; the isoelectric interval from pacing spike to QRS indicates selective His bundle capture 1 Permanent His-bundle pacing has been associated with improvements in exercise capacity, left ventricular ejection fraction, and heart failure hospitalizations, as compared with right ventricular pacing.3 Early data have suggested that either selective or non-selective His-bundle capture may have a beneficial effect with respect to cardiac resynchronization.4 However, in addition to maintaining physiologic depolarization pathways, selective His-bundle pacing may also preserve physiologic repolarization and intrinsic ST and T-wave patterns. In contrast, when depolarization is initiated in myocardial tissue (even in the case of non-selective His-bundle capture), abnormal repolarization patterns inevitably follow. Maintenance of physiologic repolarization may have antiarrhythmic benefits, and may even permit the accurate interpretation of acute ECG changes, such as ischemia or QT abnormalities, which are not generally interpretable in the setting of paced QRS complexes.6His-bundle permanent pacing has been promoted as a means to preserve physiologic conduction with ventricular pacing, with the potential to avoid pacing-induced dyssynchrony and ventricular dysfunction.