J. Chem. Theory Comput.2011, 7, 2498−250626606624) into molecular subdomains contributionsis presented and a software, DOCTRINE (atomic group DecompositionOf the Charge TRansfer INdExes) for the implementation of this novelmodel has been coded. Although our method applies to any fuzzy orto any disjoint exhaustive partitioning of the real space, it is hereapplied using a definition of chemically relevant molecular subdomainsbased on the Atoms in Molecules Bader basins. This choice has therelevant advantage of associating intra or inter subdomain contributions to rigorously defined quantumobjects, yet bearing a clear chemical meaning. Our method allows fora quantitative evaluation of the subdomain contributions to the chargetransfer, the charge transfer excitation length and the dipole momentchange upon excitation. All these global indexes may be obtained eitherfrom the electron density increment or the electron density depletionupon excitation. However, the subdomain contributions obtained fromthe two distributions generally differ, therefore allowing to distinguishwhether the contribution to a given property of a given subdomainis dominated by one of the two distributions or if both are playinga significant role. As a toy system for the firstapplication of our model, a typical  compound belonging to the merocyanine dyes familyis selected, and the first four excited states of thiscompound in a strongly polar protic solvent and in a weakly polarsolvent are thoroughly investigated.A model for decomposingthe Le Bahers, Adamo, and CiofiniChargeTransfer (CT) Excitations global indexes ( Often, the latter becomes increasinglycomplex with increasing size of the systems and new qualitative physicsbehavior also emerges. Methods for automatizing the excited stateanalysis, supported by visualization techniques7 and aimed atproviding rigorous and reproducible descriptors to measure chargetransfer (CT),13 double excitation character,16 entanglement,20 and, more generally, at disclosing phenomena that are hidden inthe standard molecular orbital (MO) picture,22 have been presented. TheoDORE22 is one of the most successful and user-friendly available methodand package in this area, being interfaced to ten different quantumchemistry codes and to a range of excited-state methods implementedtherein. Three powerful functionalities of TheoDORE have become particularlypopular, namely a “fragment-based analysis for assigning statecharacter, the computation of exciton sizes for measuring charge transfer,and the natural transition orbitals used not only for visualizationbut also for quantifying multiconfigurational character”.21Computation of the electronic structureof excited states has madeenormous strides over the past decade, due to the combined effectof the continuous, fast increase of computer power and even more soof the massive investment that has been made to develop new and evermore powerful electronic structure methods.9 In such a model, a measure of the length of aCT excitation is defined on the basis of the sole knowledge of thesystem’s ground and excited state electron densities (EDs).Within the LBAC method, the barycenters or centroids of the ED depletionand ED enrichment regions upon electron transition are computed, andthe CT length is taken as the distance between these centroids. Thenthe transferred charge is obtained by integrating either the ED depletionor the ED enrichment distribution over the whole molecular space.From the CT length and the transferred charge, the change of the dipolemoment upon electron transition is computed and compared with thatobtained from the computed ab initio ground stateand excited state wave functions, so enabling to check the accuracyof the adopted integration procedures. Our model development allowsfor decomposing the key quantities of the LBAC model into molecularmoieties contributions with the moieties being defined in terms ofa fuzzy or a disjoint exhaustive partitioning of the real space. Thechoice of subdomains is arbitrary, yet chemically meaningful moietiesshould be preferentially used to gain chemical insight into the CTprocess. Our method bears some resemblance to the fragment-based analysisin TheoDORE22 that has proved to be particularlyuseful for singling out the system fragments that mostly contributeto a given excitation and for detecting in which portions of a moleculeCT occurs. Yet, the TheoDORE method21 stronglydiffers from our presented approach being based on the concept ofa correlated electron pair and on the use of wave function for theelectron–hole pair rather than on the N-electron wave functionsof the ground and excited states. In practice, TheoDORE bases itsCT analysis on the one-electron transition density matrix, while boththe LBAC model9 and our generalizationof the LBAC model to subdomains uses the rearrangement ED, which is the difference of the excited and GS EDs. In TheoDORE,charge transfer “numbers”21 within a fragment and between fragments are customarily calculatedthrough population analysis schemes and may be somewhat or largelybasis set and computational method dependent, while in our model intra-and interfragment contributions are evaluated in the real space ofsubdomains basins and are thus much less basis set and method dependent,provided both of them are of a sufficient quality.In the present manuscript, we introduceyet another new methodable to provide a rigorous decomposition of CT excitation descriptors into atomic group (or molecular subdomain)contributions. The method represents a generalization to molecularsubdomains of the CT global indexes model developed some years agoby Le Bahers, Adamo, and Ciofini (hereafter LBAC model).10 therebyproviding experimental chemists with useful insights to design push–pullcompounds with targeted properties. Push and pull systems, consistingof an electron donor (D) and of a covalently connected electron withdrawing(A) group have been largely investigated owing to their intense, solvatochromic,optical transitions.26 However, the simple mechanism of the formation of an excited statewhere an electron is transferred from the D to the A to form a [D+A–]* excited state is just an ideal situation.27 Excitations may, in reality, be local or delocalizedin character, they may not take place necessarily from the D to theA, and besides that, the effective transferred charge may be muchlower than the ideal value of one for single-electron excitation processes.28Using thelength and magnitude of charge transferred, the LBACmodel was devised to screen on a qualitative basis push–pullcompounds belonging to diverse chemical families,Photoinduced electron-transfer (PET)is a key mechanism of variouschemical, physical, or biological processes, all having intensivelyscrutinized applications areas, including light-to-chemical energyconversion, molecular photoelectronics, photocatalysis, or photosynthesis.The ability of tuning and/or improving charge transfer and of identifyingthe actors playing the major role into the electron-transfer mechanismsis clearly a prerequisite for designing optimized systems.Extensionof the LBAC model to a subdomain representation, enablingus to distinguish and quantify the local intrasubdomain effects fromthe synergic or anti-synergic coupled effects of all subdomain pairson the global CT indexes, may hopefully provide further precious insighton the PET processes.3N) and electronacceptor A (CO) groups are separated by a conjugated system, enablingthe electron drift between these two components. Its electronic structuremay be envisaged as a resonance hybrid between twolimiting structures, a noncharge-separated one  and a charge-separated structure  pm b to a quinonoid pro-aromatic (BM)q structurein protic solvents, the opposite being generally true in apolar solvents.33As a “toy” system for thefirst application of ourmethod, we selected a typical  compound belonging to the merocyanine dyes family,namely the 1-methyl-4-[(4-oxocyclohexadienylidene)ethylidene]-1,4-dihydropyridine,also called Brooker’s Merocyanine (BM), taken in its trans-deprotonatedform 1. Inthiic form) 1.30 Yet,33 In this work, the trans-deprotonatedform of the BM molecule was considered in various solvent media characterizedby a wide spectrum of dielectric constants and by making use of eitheronly an implicit (SMD) solvent model or the same SMD model but witha further inclusion of explicit solvent molecules.We have recently performeda systematic, comprehensiveDensityFunctional Theory (DFT) and Time Dependent Density Functional Theory(TD-DFT) study of the photoinduced CT processs in the BM compound,as a function of the various BM excited states and of the adoptedsolvent model.qCT upon excitation, the CT excitationlength, DCT, and theresulting change in the magnitude of the molecular dipole, |ΔμCT|). All these global indexes were calculatedthrough our implementation of the standard LBAC model. Additionally,LBAC indexes were also put in relation with the evolution of localfeatures that characterize—upon vertical excitations—eitherthe chemical bonds or the electron delocalization in the various BMmoieties.33The aim ofthis recent study was to exploit the possibility toreach a suited manipulation of the CT process thanks to the quitedifferent nature, in a given solvent, of the first four BM excitedstates, and to the significant alteration of the excited states naturethat may be induced by choosing among 24 different solvent models.Electronic transitions were first characterized in terms of LBAC globalindexes  and of the vertical excited state n, respectively,the ED rearrangement due to the electronic transition Sn ← S0 is given byR3  ishereafter simply written as Δρ(r)). By defining ρ+(r) and ρ–(r) as being equal to |Δρ(r) | if Δρ(r) is, respectively,greater or smaller than zero, and equal to zero if Δρ(r) is, respectively, smaller or greater thanzero, i.e.:qCT upon excitation as the following quantity:Named as qCT will range between 0 and 1 for a one-electronexcitation, being close to 1 for a nearly ideal one-electron transferand even much less than so in real situations. The centroids or thelocations of the poles of the positive and negative transferred chargeare given byDCT, is obtained fromμCT,is calculated asabinitio dipole moment magnitudes computed for the ground andthe excited state n gives an estimate of the integrationaccuracy in The norm ofthe dipolemoment change between the ground and theexcited state, positively charged relative to the GS) and negative Δρ values to the regionsthat increase their electron concentration (hence becoming negatively charged relative to the GS).It is important to note thatin R3 in subdomains Ω, qCT as caused by a sum of subdomainscontributions. Notice that the equivalence of the sums of subdomaincontributions calculated by integrating either ρ+(r) or ρ–(r) over the whole set of Ω does nothold true for the separate subdomain contributions. In general, ∫Ωρ+(r)dr ≠ ∫Ωρ–(r) dr and the following subdomain quantitiesmay be defined as follows:By assuming afuzzy or a disjoint exhaustive space partitioning of qCT(Ω) to appreciate whether a subdomainis more responsible of creating one or the other of the two poles.Note also that ∑ΩΔqCT = 0 and that the following inequalitiesholdr) values.qCT, also DCT may be conveniently writtenin terms of subdomain contributions,dCTΩ, different from DCT, is a 3-component vector. Hence, we introducethe correspondingvector for the whole system, dCT, whose componentsare given by a sum over the corresponding subdomain components:dCT, jΩ(Sn ← S0) length relativeto the DCT length. Anegative sign of gCT, jΩ means that dCT, jΩ is oppositely directed to (R+ – R–)j. Also usefulis to introduce the direction cosines of (R+ – R–), αj , i.e.:dCT to (R+ – R–) and where aj is the angle between the j axis and the (R+ – R–)vector.Likewise μCT∥(Sn ← S0), is given by the product of DCT and qCT and itsdecomposition in subdomain contributions is not straightforward fortwo main reasons. First, one has to introduce a separate decompositionfor each dipole moment change component:μCT, j would be possible, e.g. by assumingto assign to each subdomain half of its subdomain pairs contributions,this partitioning remains arbitrary, and we thus prefer to avoid inthis manuscript any subdomain decomposition based on Mj,+ having as diagonal elements the single subdomain contributions dCT, jΩ·qCT+(Ω) and as out-of-diagonal elements the mixed terms dCT, jΩ·qCT+(Ω ′) are of some interest and are worth investigating.For instance, each μCT, j may be easily decomposed into an intrasubdomainscontribution, μCT, jintra,+, and in an intersubdomains counterpart, μCT, jinter,+, as follows:Mj,+ matrix is dimensioned nsub times nsub where nsub is the numberof considered subdomains. Clearly an analogous Mj,– matrix may also be defined, havingas diagonal elements the single subdomain contributions dCT, jΩ·qCT–(Ω) and as out-of-diagonal elements the mixed terms dCT, jΩ·qCT–(Ω ′). Both matrices Mj,+ and Mj,– have the property to reproduce the ∥μCT∥ value from either their μCT, j+ or μCT, j– vector components, or equivalentlyby summing up either all the Mj,+ or all the Mj,– matrix elements , represent the subdomain Ωi internal contribution and the subdomainΩiΩk pair contributions, respectively.As indicated in elements 15. Yet, μCT∥ may be realized if only thedependence of ∥μCT∥ on qCT is taken into account. In such a case, a quite simple expressionresults:μCT∥ but in terms of two different subdomaindecompositions, one based on qCT+(Ω) andthe other on qCT–(Ω). It is of someinterest to compare these two alternative subdomain decompositionsof ∥μCT∥,one emphasizing the role of qCT+(Ω) andthe other that of qCT–(Ω). Both decompositionsclearly have a chemical significance.A much simpler subdomaindecomposition of ∥34 zero flux condition for definingthe subdomains Ω, i.e.,rs is any point onthe subdomain surface boundary S and n(rs) isthe normal to the surface at rs, enables us to associate the CT indexes subdomaincontributions to atoms or groups of atoms rigorously defined throughquantum mechanics.34As stated earlier, 35 (atomic group DecompositionOf the Charge TRansfer INdExes), that, as a first step, calculatesall the QTAIM atomic basin contributions to the CT indexes. In practice,at this stage, Ω is any atomic basin of the molecule in the 36 of the groundand of the excited state n of the molecule beinginvestigated, along with the file (in .sur format) containing thesurface boundary information on all QTAIM atomic basin Ω ofthe molecule in the ground state. The data in the .sur file are computedby a previous PROMEGA calculation (PROMEGA is one of the codes ofBader’s AIMPAC95 package)37 thatevaluates the boundaries of all atomic basins in the GS molecule.Integral properties of the QTAIM basins may also be calculated atthis stage if of interest for relating them to the computed CT indexes33 and to their atomic group decomposition. Clearly,in such a case, the atomic boundaries and the integral propertiesof the excited state need also to be computed to evaluate the integralproperties changes upon electron transition. DOCTRINE evaluates ∥μCT∥(Sn ← S0) from μCT∥(Sn ←S0) from its atomic basin Ω contributions with thevalue computed by the GAUSSIAN 16 code permits to evaluate the accuracyof the numerical integration. Since this is performed for all QTAIMatoms in spherical coordinates and on an atomic centered grid, theresulting accuracy is noteworthy, provided a suitable number of angularand radial points is used in the Gaussian quadrature integration procedure(see μCT∥(Sn ← S0) values obtained from qCT+ (Ω) or qCT– (Ω) data or the qCT values obtained from + or ρ– distributions.Based on the just illustrated premises, we have written a code,DOCTRINEduresee . Furthernsub atomic group contributions, each atomicgroup being composed by a suitably selected disjoint subset of theatoms of the investigated molecule. Clearly, the so defined nsub molecular subdomains need to collectively include allatoms of the molecule.Once the QTAIM atomic basin Ω contributionsto the CT indexeshave been calculated, DOCTRINE combines and gathers them into theselected 4 and in water, as representativeexamples. CCl4 is a weak polar nonhydroxyl solvent (dielectricconstant ε = 2.23), while water is a strong polar protic solvent(ε = 78.4). In the case of water, two explicit solvent (ES)molecules have been considered, since the formation of hydrogen bonds(HBs) between the carbonyl end of the BM molecule and the hydrogenatoms of water solvent molecules has been suggested to present stronginvolvement in the electron transition properties. Furthermore, thecrystallized BM compound includes structural solvent molecules.39 This explicit solvent model for BM in water is hereafter referredto as BM + ES2. For both solvent cases, the following fourBM moieties were considered in our analysis: the carbonyl oxygen atom,the 6-Carbon Membered Ring plus its four linked H atoms, the two Catoms of the Central Bridge plus their two linked H atoms and theMethylPyridine ring along with its linked 4 H atoms. In the following,these four BM moieties (≡ BM Ω subdomains) will be calledas O(CO), 6CMR, CB and MePy, respectively. The .wfn files for thetwo ground states and eight excited states investigated in the presentstudy have been taken from our previous work on the BM molecule excitations.33 Quantum chemical calculations were carried outthrough the Gaussian 16 software package and using DFT and TD-DFTprocedures. Geometry optimization [S0 or singlet groundstate (GS)] and TD-DFT treatment for the vertical excited states  were allperformed using the long-range corrected (LRC) Coulomb attenuatedB3LYP xc functional CAM-B3LYP40 with the cc-pVDZ basis set and by including solvent effectsthrough the implicit SMD solvation model.41For this study of the atomic groups decompositionof the LBAC globalindexes, we have used the GS and the first four excited states ofthe BM molecule in CCl2 for the water model, besidesconsidering the implicit dielectric effect of solvent through SMD,two explicit water molecules were initially placed in the neighborhoodof the C=O group and left totally free to geometrically relaxduring the geometry optimization step. The local energy minimum natureof the calculated structures was confirmed from their (harmonic) vibrationalanalysis implemented in the Gaussian 16 software package (no imaginaryfrequency).In the case of BM + ES2 in water system, the adopted .wfn filesfor both the GS and the excited states were those obtained by restrainingexcitations and charge transfer to the BM molecule only . We had howeverto verify that no CT occurs to the explicit solvent molecules alsowhen a QTAIM space partitioning is adopted. Indeed, the QTAIM netcharge on the two explicit solvent water molecules has been alwaysfound to be below ±5 × 10–3 electronsfor all investigated cases. These results enable us to restrain thedecomposition of global CT indexes to contributions from subdomainsformed by atoms of the BM molecule only also for the BM + ES2 in water system.The global CT indexes, along with their atomic groupdecomposition,have been obtained from the DOCTRINE code (see previous paragraph).For the BM + ESi.e., the atomic boundaries determination, needs however to be performedonly for the ground state of a system, regardless of the number ofits investigated excited states.Atomic boundaries for the ground states weredetermined by PROMEGAcode using typically 6144 angular points . Evaluation by the DOCTRINE code of the atomiccontributions to the global CT indexes used 200 radial points in theGaussian quadrature integration procedure outside the so-called betasphere and the same angular points of the previous determination ofatomic boundaries by the PROMEGA code. The evaluation of atomic contributionsto the global CT indexes is about 2 orders of magnitude faster thanthe atomic boundaries determination, and it is therefore very fast. The CPU more demanding step, μCT∥ = ∥μCT∥DOCTRINE code – ∥μCT∥Gaussian 16 code for all investigated excitations. Δ∥μCT∥ is the difference of the ∥μCT∥ value obtainedfrom the separate atomic or atomic group contributions to ∥μCT∥ evaluated bythe DOCTRINE code and that calculated directly by the Gaussian-16code. The largest Δ∥μCT∥ difference reported in 3 ← S0 excitation of BM + ES2 in water. Typically, Δ∥μCT∥ values are found to be one order or even2 orders of magnitude smaller. qCT upon excitation calculatedby integrating either ρ+(r) or ρ–(r) over the whole set of atomic or atomic groups subdomains of theBM molecule, for all studied excitations. The differences betweenthe qCT values obtainedusing one or the other of the two densities, also reported in qCT values. Such differences never exceed0.006 e– and in most cases are even 1 to 2 ordersof magnitude smaller.n (n =1–4) of the BM+ES2 in water and the BM in CCl4 systems, respectively. The decomposition into subdomain contributionsof one of these global indexes, namely the amount of the transferredcharge qCT whose valueis reported in e– over the “+” signin each of the left panels, is instead shown in the right panels ofboth figures. The qCT decomposition is presented both in terms of qCT+(Ω) and qCT–(Ω) contributions.Furthermore, the difference, ΔqCT(Ω), between the qCT+(Ω) and the qCT–(Ω) contributionsis also displayed. A of thesum of only positive ΔqCT(Ω) values, qCT(Ω) values; see n, evaluated bythe DOCTRINE code and reported in Debye (D); (ii) the CT excitationlength, DCT, given inÅ, and (iii) the locations of the positive and negative centroidsof the transferred charge upon excitation. The location of centroidsis indicated by purple balls with a positive or negative sign identifier.As expected, in almost all excitations the electronic charge is transferredfrom the D to the A moiety of the BM molecule  a reversed CT occurs, from theA to the D moiety of the molecule. With the exception of the S4 ← S0 excitation, the change of the dipolemoment caused by the electronic transition is definitely larger inthe protic water solvent (both when implicit and explicit water solventmodels are adopted)33 than it is in theweakly polar CCl4 solvent. Rather than to an enhanced chargeseparation qCT, the larger∥μCT∥is due to the much larger charge excitation length in the protic solvent,that is to the largely enhanced spatial separation of the positiveand negative charge centroids  states 2 and 3. In other words,states 2 and 3 of BM + ES2 in water need to be associatedand compared, respectively, to states 3 and 2 of BM in CCl4. The shrinking of the charge excitation length in the weakly polarsolvent, relatively to BM + ES2 in water, is particularlyevident for the S1 ← S0 transition (0.40Å rather than 3.85 Å for BM + ES2 in water),which exhibits also a reversed sign, as mentioned earlier.In their leftpanels, qCT(Sn ←S0) in either qCT+(Ω) or qCT–(Ω) contributions show the differentroles played by the various subdomains in determining the amount ofthe transferred charge, according to the investigated system and electronicexcitation. In the BM + ES2 in water, all transitions arecharacterized by the MePy subdomain, providing the most relevant qCT–(Ω) contribution consistentlywith its role of acceptor, except for S4 ← S0 excitation where the qCT–(Ω)contribution from the 6CMR prevails. On the other hand, in the sumof the qCT+(Ω) values, also yielding qCT, different subdomains playthe major role, depending on the transition. The carbonyl oxygen dominatesthe qCT+(Ω) sum in the case of the excitationwith the largest qCT,∥μCT∥,and DCT magnitudes (S3 ← S0), while it is the 6CMR that playsthe leading role in the other three transitions. The reconstructionof qCT in terms of twoalternative and different reconstructions enables us to discriminatethose subdomains that concur to cause the charge separation owingto a significant increase or decrease of their electron populations,from those that marginally change their electron populations and contribute to the charge separation because of a Δρ(r) polarization in their subdomain, withportions of the subdomain characterized by significantly positiveΔρ(r) values and other portionsby significantly negative Δρ(r) values. The former are characterized by comparatively large |ΔqCT(Ω)|values, while thelatter have qCT+(Ω) and qCT–(Ω) contributions that tend to compensate eachother, leading to comparatively small or very small |ΔqCT(Ω)| values. As a consequence,while MePy behaves in general as a true acceptor, hence having largelynegative ΔqCT(Ω)values, the role of the true donor, which should have largely positiveΔqCT(Ω) values,is less evident and played either by the carbonyl oxygen (S3 ← S0 and S4 ← S0)or by both the 6CMR and the carbonyl oxygen but in larger measureby the former (S1 ← S0 and S2 ← S0). The CB subdomain usually seems to playa dual role in causing charge separation, polarizing itself in responseto the excitation, exhibiting not negligible qCT+(Ω) and qCT–(Ω) contributionsbut generally small and either small positive or small negative ΔqCT(Ω) values. This behaviorcomplies with the carbon bridge being a conjugation entity covalentlyconnected to both the D and A moieties of the BM molecule and supportingthe electron transport between them. The major or minor weight ofthe two possible situations described above may be easily appreciatedby examining the ratio of A to qCT , while inthe former transition the MePy and in particular the 6CMR subdomainshave an evident dual behavior, with part of their basins acting aselectron acceptors and other not negligible portions of their basinsas electron donors. Note that the trend of the qCT and ∥μCT∥ values. Forinstance, the largest qCT and ∥μCT∥values occur for the S3 ← S0 excitation,yet the highest 2 ←S0 transition. Indeed the location of the centroids ofthe transferred charge clearly indicate that the CT for the S3 ← S0 excitation is essentially taking placewithin the 6CMR region where, though with clamped nuclei, a switchfrom the benzenoid (GS) to the quinonenoid BM electron structure likelyoccurs  valueof 0.043 e– and, consequently, a smaller value forthe total ΔqCT relativeto the S2 ← S0 transition where the 6CMRbehaves instead as a clear electron donor. Our reasoning is also supportedby the more than doubled decrease of the C–C delocalizationindex (DI)42 of the central bond of thecarbon bridge found for the S3 ← S0 relativeto the S2 ← S0 transition  = −0.17 vs  = −0.07. The delocalizationindex measures the number of electron pairs shared between two atoms,and it is close to 1 for a CC single bond and it reaches a value ofalmost 2 for a double CC bond. A decrease of the DI for the centralCC bond of the carbon bridge implies an increase of the quinonoidrelative to the benzenoid BM structure, upon excitation  or for S2 ← S0 (D–D–D–A) excitations.Features and trends of the global CTindexes have been briefly commented above in order to analyze thefurther information that may be obtained from their decompositionin subdomain contributions. The decomposition of A to qCT 9, that ayoccurs 1.Such ation see 1. As a fansition 1. The S4eresult 2 of an aeresult 1 whereasn (n = 1–4) vertical excited states of BM in CCl4 incomparison to those already discussed for the water solvent case.The 4 amounts to just 0.04, 0.33, 0.48, and 0.46,so it is lower or much lower than for BM + ES2 in water,except for the S4 ← S0 transition thatis quite anomalous for this latter solvent model. The generally lower μCT∥ and DCT values for correspondingstates in the BM in CCl4 vs BM + ES2 in waterare but the consequence of the general D and A dual behavior of subdomainsin the former system. The case of the S1 ← S0 excitation (qCT(Ω) values.This is the only case, among the eight here investigated, where theMePy subdomain has a totally marginal role as a donor (ΔqCT = −0.008 e–) notwithstanding the significant, yet almost equal in value, qCT+ and qCT– contributions. The negligibleΔqCT(MePy) value,along with the carbonyl oxygen playing the prevalent role of weakacceptor rather than strong donor (ΔqCT(O(CO) = −0.005, μCT∥ and DCT values have instead well separate donorand acceptor regions, with D–A–A–A (S2 ← S0) or D–D–D–A (S3 ← S0) D/A patterns (4 ← S0) transitionhas the lowest ∥μCT∥ and DCT values among the excitations with direct CT and, not unexpectedly,has an almost alternate D/A pattern, namely A–D–A–A,with (O(CO) playing the role of the acceptor rather than donor. Thischange from the usual role for the O(CO) corresponds to an enhancedweight of the benzenoid rather than of the quinonic BM structure uponexcitation. This is corroborated by a 0.03 e– increaserather than a decrease of the electron population of the oxygen atomand a small 0.01 increase, rather than a clear decrease, of the DIvalue of the carbon bridge central CC bond in the excited state.Let usexamine now the Scitation 2 is partμCT∥ (Sn ← S0), between the ground and the excited state n. In the Figure and μCT∥ decomposition is performed according to μCT∥ on qCT is taken into account. The total dipolemoment change for each excitation is represented by a pale green barand both the μCT+(Ω) and the μCT–(Ω) contributions to the total dipole momentchange are shown for all four subdomains Ω considered in ouranalysis.2 in water,the ∥μCT∥reconstruction in terms of μCT+(Ω) isdictated by the carbonyl oxygen and by the 6CMR contributions . The 6CMR contributionis the largest one for the first two excited states, whereas it isthe carbonyl oxygen that dominates the third excitation with a 61%contribution to what represents the largest ∥μCT∥ value (20.8 D) among the eightinvestigated cases. Not unexpectedly, the decrease of the carbonyloxygen electron population along the series of the first three excitedstates  parallels the increase(from 18 to 21 to 61%) of its μCT+(Ω) contributionto ∥μCT∥.The fourth excited state of BM+ES2 in water has a small∥μCT∥value (3.4 D) with comparable μCT+(Ω) contributionsfrom the four subdomains. The ∥μCT∥ reconstructions in terms of μCT–(Ω) contributions differ from those using the μCT+(Ω) ones, as expected. The first twoexcited states are governed by the MePy contribution amounting to61 and 82% of the total, while in the third state MePy exhibits stillthe largest contribution (42%), but also the other 3 subdomains givesubstantial and similar to each other contributions summing up tothe remaining 58%. Therefore, for the excitation with the largest∥μCT∥value, the μCT+(Ω) reconstruction is evidentlygoverned by the carbonyl oxygen, whereas all four subdomains concurwith relevant weights to the ∥μCT∥ reconstruction in terms of μCT–(Ω), as clearly anticipated by the associated qCT decomposition using qCT–(Ω). The ∥μCT∥ reconstructions for BM inCCl4 look different from those of the BM+ES2 in water. Yet, those for states 2 and 3 are qualitatively similarif the mentioned interchange between states 2 and 3 is taken intoaccount. The first excited state exhibits similar and small contributionsfrom all subdomains in both the μCT+(Ω) and μCT–(Ω) reconstructions except thecarbonyl oxygen subdomain that gives an almost zero contribution inboth sums. For the fourth excited state, the 6CMR dominates the ∥μCT∥ reconstructionin terms of μCT+(Ω), while in that basedon μCT–(Ω) the 6CMR still providesthe largest contribution, but both the CB and the MePy moieties arealso contributing in a significant way.In the first three excited states of BM + ESx component, dCT, xΩ, of the excitation lengthvector, DCT, for the first Sn (n = 1–4) vertical excited states of BM + ES2 inwater and of BM in CCl4. Only the x component, dCT, xΩ, of the atomic groups contributionto the excitation length vector DCT is shown in the x axis is almostcollinear and, for all cases but one (S1 ← S0 of BM in CCl4), antiparallel to DCT. The degree of collinearityis measured by αx (see x-axis direction cosine of DCT= (R+ – R–), which islisted in the x axis is taken as directedfrom the left to the right of the molecule whereas R+ – R– is almost oppositelydirected if the locations of the centroids of positive and negativetransferred charge reflect a photoinduced CT from the O(CO) and the6CMR to the MePy moiety of the BM molecule. Apart S1 ←S0 of BM in CCl4, this is indeed the commonsituation in the eight reported cases, and therefore, the x component, dCT, xΩ, of the excitation length vector, DCT, is generally negative the gCT, xΩ value multiplied by 100 expresses the percentage contribution ofthe subdomain to the observed excitation length. In the first twotransitions of BM + ES2 in water, it is the MePy subdomainthat governs DCT while it is the carbonyl oxygen that takes this role for the two subsequentexcitations  being −0.57 and −0.92). Withthe exception of S4 ← S0 the 6CMR appreciablyconcurs to increase the excitation length, but in the case of theS4 ← S0 it clearly contrasts it. Notunexpectedly, this behavior is associated with the 6CMR behaving moreas an A rather than a D for this excitation  and, in but one case, allcontrasting the global excitation length. In the case of BM in CCl4, a different subdomain plays the major role for each excitation,namely CB for the first ,O(CO) for the second one, MePy for the third transition , and finally,the 6CMR for the fourth one .y αx see 13, i.e.,negative 4. The co DCT see 12, are acitation 1. The cox-component of the μCT vector into its intra-subdomains contribution, μCTintra, and its inter-subdomains contribution μCTinter for the first Sn (n = 1–4) vertical excitedstates of BM+ES2 in water and of BM in CCl4.The decomposition of the μCT vector is afforded through μCT∥on both qCT and DCT and not just qCT The other two μCT vector components  have comparatively negligible values due to the fact that the excitationlength vector DCT is almost parallel or antiparallel to the x axis in all excitations  transitions are reported in the Supporting Information, Tables S1–S3.As the excitation length vector DCT is almost parallel or antiparallel to the x axis in all excitations, the elements of the matricesof the components y and z of the μCT vector have values thatare, in general, comparatively negligible in magnitude relative tothose of the x component matrix and are thus notreported. The sum of the elements on the diagonal of the matrix Mx,+  (those with pink background) correspondsto the intra-subdomains contribution, μCTintra, to μCT, x+  while the sum of the out of diagonal elements (those with lightblue background) corresponds to the inter-subdomainscontribution, μCTinter, to μCT, x+   15. AnalyMx,+ and Mx,– matrices relative to the S4 ←S0 transitionin BM+ES2 in water, it is easy to observe that the dominantmagnitudes of the μCT, xinter,+ and μCT, xinter,– vectors  and, among qCT+(Ω), in particular qCT+(6CMR) and those ofthe fourth row, involving dCT, xMePy and, among qCT+(Ω), again qCT+(6CMR), in particular. Other mixed terms inthe second and third row are found to oppose the observed μCT and their total sum amounts to 1.25and 1.10 D for Mx,+ and Mx,– relative to μCT, xinter,+ and μCT, xinter,– magnitudes of −2.8 and −3.3 D , so both the diagonal and the out of diagonal elementsof matrices Mx,+ and Mx,– play a relevantrole in determining the dipole moment change upon electron transition.Concerning Mx,+, arethe intrasubdomain dCT, x6CMR·qCT+(6CMR) and the intersubdomain dCT, xMePY·qCT+(6CMR) contributions that mostly favor thedipole moment change ·qCT+(6CMR), with a value of 1.060 D, that mostly opposes the dipolemoment change. Concerning Mx,–, the terms of the second row involving dCT, x6CMR and qCT+ and those of the fourth row involving dCT, xMePY and qCT+ play the majorrole in favoring the observed dipole moment change. Other contributionsare less relevant in favoring the dipole or act to oppose it to variousextents, the most effective, with a value of 1.060 D, being dCT, xO(CO)·qCT+(6CMR).In the case of the SMx,+ and Mx,– matriceslistedin Tables S1–S3, for theremaining six investigated electron transitions, have a similar purposeand may be analyzed following the same lines sketched above for theonly cases of the S4 ← S0 transitionin the systems BM+ES2 in water and BM in CCl4. For the sake of space, their discussion is therefore not reported.Just note that the transition with the largest dipole moment change has all the Mx,+ and Mx,– elements bearing the same sign, thereby indicatingthat all intra- and intersubdomain contributions jointly cooperateto yield the observed dipole moment change.The 9 into molecular subdomains contributions, anda software package for the application of the method has been coded.Analogously to the LBAC original model, the subdomain decompositionof the CT indexes is made in the real space, using the rearrangementelectron density given by the difference between the electron distributionin the excited state and that in the ground state. Working in realspace has the special advantage that the intra- and interfragments contributions to the CT indexes, analogousto their global sums, are much less basis set and method dependent,provided that both the basis set and the quantum mechanical adoptedmethod are of a sufficient quality.We have presented a new method for decomposingthe Le Bahers, Adamo,Ciofini (LBAC) Charge Transfer Excitations global indexesintra- or intersubdomain contributions to rigorouslydefined quantum objects, yet bearing a clear chemical meaning.Our method applies to anyfuzzy or to any disjointed exhaustivepartitioning of the real space. However, using a definition of chemicallyrelevant molecular subdomains based on the Atoms in Molecules Baderbasins has the important advantage of associating The developed method allows for a quantitative evaluation of thesubdomain contributions to the charge transfer, the charge transferexcitation length, and the dipole moment change upon excitation. Allthese global indexes may be obtained either from the electron densityincrement or the electron density depletion upon excitation. However,the subdomain contributions obtained from the two distributions generallydiffer, therefore allowing one to distinguish whether the contributionto a given property of a given subdomain is dominated by one of thetwo distributions or if both are playing an important role.toy system for the first application of ourmodel, a typical compound , belonging to the merocyanine dyes family has been selectedand the first four excited states of BM in a strongly polar proticsolvent and in a weakly polar solvent have been scrutinized. It isremarkable to note how the subdomain contributions of the transferredcharge and of the CT excitation length are able to reveal the causesbehind the distinct features of the investigated excitations. Theglobal CT indexes have often underlying chemical motifs that wouldbe hardly possible to quantify or to even imagine without decomposingthem in suitable concurring or opposing contributions. Since the subdomaincontributions of the transferred charge and of the CT excitation lengthjointly concur also to determine the change in the dipole moment uponelectron transition, the subdomain contributions patterns representa precious and chemically insightful representation of the photoexcitedtransitions.As a In overall, we hope that our method may provideanother usefuland distinct tool among the many that have been already developedto help practitioners in the field of light-driven charge transferprocesses and that the subdomain decomposition of the CT indexes mayserve to further deepen our detailed understanding of these processes.


We foundthat a mechanical rotation of the sample does not alter polarization-resolvedRaman spectra, which can only be explained by introducing an antisymmetricRaman tensor, frequently used to describe Raman optical activity (ROA).Raman spectra obtained under circularly polarized excitation demonstratethat 1T-TaS2 indeed shows ROA, providing strong evidencethat 1T-TaS2 is chiral under the used conditions of laserexcitation. Our results suggest that ROA may be used as a universaltool to study chiral properties of quantum materials.Measurements of opticalactivity can be readily performed in transparentmatter by means of a rotation of transmitted light polarization. Inthe case of opaque bulk materials, such measurements cannot be performed,making it difficult to assess possible chiral properties. In thiswork, we present full angular polarization dependencies of the Ramanmodes of bulk 1T-TaS Such measurements can be readilyperformed in the case of transparent materials but cannot be appliedto bulk metals because no light is transmitted. This fact makes itdifficult to optically assess the chiral properties of complex systemslike the charge density wave (CDW) compounds 1T-TaS2 or1T-TiSe2.2 In this work, weprovide evidence for the optical activity of 1T-TaS2 byusing polarization-resolved Raman spectroscopy.Since the first observationof optical activity of quartz by Aragoin 1811,2 undergoes temperature-dependent phase transitionsaccompanied by periodic lattice distortions related to charge densitywaves.5 It adopts the following four CDW phases: an undistorted, incommensurate CDW (ICCDW), nearly commensurate CDW (NCCDW),and completely commensurate CDW (CCDW). The electronic and surfacestructure of 1T-TaS2 in different phases has been extensivelystudied by angle-resolved photoemission spectroscopy (ARPES) and scanningtunneling microscopy (STM) techniques.91T-TaS2 can persistin mixed-metastable states11 or can be used as reversiblememristor material.13 An intriguing property of 1T-TaS2 is the presence of a hidden state, which can be enabled byan ultrafast light pulse15 and which can alsoinfluence the CDW stacking in the out-of-plane direction.16 A recent work shows that changes in the CDWstacking can even be caused by incoherent white light illumination.17 These findings highlight that light illuminationcan directly influence the CDW properties of 1T-TaS2, andcurrently it is not established how results obtained by optical spectroscopyare affected by these effects. Our research demonstrates that Ramanscattering in bulk 1T-TaS2 is a complex phenomenon, whichmust be analyzed in terms of the influence of light illumination,resonant effects, and optical activity.Despite these phase transitions, 1T-TaS2 (hqGraphene) crystals were examined bymeans of Raman spectroscopy using a Horiba T64000 spectrometer. Low-temperaturemeasurements (5 K) were performed using a liquid helium flow cryostat(Oxford Instrument Hires2). Lasers with wavelengths of 442, 532, 633,and 785 nm were used as an excitation source. The laser beam was focusedby a 50× objective to an ∼1 μm spot size, and thepower of ∼300 μW was chosen to avoid heating effectsand damage of the sample.Bulk1T-TaSSupporting Information). In order to collect spectra dependent on thesample rotation, a half-wave plate was mounted above the cryostat,providing conditions corresponding to a sample rotation experiment(see Supporting Information).For linear polarization-resolved Ramanexperiments, incident lightpolarization was fixed. A half-wave plate was mounted in front ofthe CCD camera, followed by a linear polarizer, which was set parallelto the incident light polarization axis (see Supporting Information). This setup allowed for collecting Raman spectra in four differentconfigurations of excitation/detection: σ+/σ+, σ+/σ–,σ–/σ+ and σ–/σ–.Circularlypolarized incident light was obtained by placing a quarter-waveplate behind the first polarizer, allowing for excitation by right-(σ+) or left-handed (σ−) polarized light. A secondquarter-wave plate was placed in front of the second polarizer beforethe CCD camera  are shown in –1 were detected.It is commonly accepted that the observed phonon bands originate fromfolding of the Brillouin zone as 1T-TaS2 undergoes CCDWphase transition.20 As can be seen, for different excitation energies,some lines do not significantly change their intensities (228 cm–1), however, most of them show observable differences.A notable example is the line at 114 cm–1 that iscompletely attenuated for 633 nm excitation. The doublet with linesat 243 and 248 cm–1 changes its intensity ratio.Under higher (lower) excitation energy the 243 cm–1 (248 cm–1) line is more pronounced. The linesat 256 and 298 cm–1 are most intense under 532 nmexcitation, whereas the 292 cm–1 line is more pronouncedfor lower energy excitation (633 and 785 nm wavelengths). The linesat 256, 283, and 307 cm–1 are very weak when excitedwith a 785 nm wavelength laser. –1, excited with 442, 532, 633, and 785 nm wavelengthlasers.Unpolarized Raman scattering spectra of bulk 1T-TaS22 When the excitationenergy is tuned to a maximum in the material’s joint densityof states, a resonant Raman term must be taken into account, resultingin an enhancement or attenuation of a particular mode. For a betterunderstanding of resonant effects, one must refer to the full quantumRaman scattering model,24 where the Raman scattering intensity depends on the excitation energy.Changes in Raman mode intensities under differentexcitation energiesshown in 2 in the CCDW phase has a large number of bandsin the Brillouin zone for the energy range covered by the excitationenergies applied in our experiments (1.6–2.8 eV).26 These energies are well abovethe reported values for band gaps of about 100–400 meV.28 By increasing the energy of theexcitation laser, one covers many subsequent energy states, whichare located close to each other. This results in the observed effects,that is, attenuation, enhancement, increase or decrease, of the Ramanintensity of particular modes, as reported for other TMDCs.22 Additionally by selecting energy states with different symmetries,the polarization properties of Raman modes can also be altered, whichwill be discussed later.1T-TaSIn order to further study resonanteffects and the symmetry ofRaman modes we performed polarized Raman measurements both in scatteredlight polarization rotation and in sample rotation configuration.The obtained Raman spectra are shown in T = 5 K). Green (red) circles show data obtainedfor 532 nm (633 nm) wavelength excitation. The angle θmax for which the plot is the most intense was obtained by fitting thefunction: I = A + B cos2(θ – θmax), shown asgreen and red solid lines in max. Thereare lines that preserve the main axis of polarization of the laser(0°) and lines with the main axis rotated by ∼60°,∼120° or ∼90°. Almost all lines polarizedin 0° direction are fully polarized, whereas the polarizationdegree of lines showing other polarization angles differs from ∼0.5to ∼0.7. Interestingly, the 248 cm–1 lineis fully polarized when excited with green light, but weakly polarized,when excited with red light. Similarly, the 243 and 292 cm–1 lines are less polarized when excited with a 633 nm wavelength laser.Moreover, the polarization angles θmax depend stronglyon the excitation energy for a number of lines . For both 134 and 277 cm–1 lines, the main axis changes significantly (by about 40°).Angular intensity plots obtained as a function of the scatteredlight polarization angle (with fixed incident light polarization)using different excitation sources (532 and 633 nm wavelength lasers)are shown in 2 in the visible range, we resonantly excite many different electronicstates at the same time. These states may have different symmetryproperties and, consequently, angular plots consist of a mixture ofdifferent polarization directions, which results in a lower polarizationdegree. However, strongly polarized lines generally do not changetheir main axis angle, especially lines polarized in 0° direction(see Supporting Information).These results most probably stemfrom resonant effects. By changingthe excitation energy, we can select states with different symmetriesand therefore create resonant conditions with different symmetries,as shown in refs , and 29.2 in the CCDW phase belongs to the Ci3 symmetry point group andhas two types of Raman active modes: Eg and Ag. On the basis of the semiclassical model, the Raman mode intensity I can be written as30ei and es stand for incidentand scattered light electricfield unit vectors, respectively, and R is Ramantensor for a given mode. Superscript t of es denotes the transformation from a column vector to a row vector.Applying appropriate Raman tensors and electric field unit vectorsallows us to model the Raman intensity dependency on the scatteredlight polarization rotation and sample rotation (see Supporting Information). According to this theory, all linesshould be linearly polarized. In the case of scattered light polarizationrotation, Ag modes should preserve incident light polarizationregardless of the sample rotation, whereas the main axis of the Eg modes should depend strongly on the crystal alignment withrespect to the incident light polarization. This means that Ag modes should be polarized along 0° and should be attenuatedin cross-polarization configuration, while Eg modes shouldbe detectable both in co- and cross-polarization configuration. Assuminga Ci3 crystal point group,we assigned Ag and Eg symmetry to all detectedRaman modes, as shown in g andEg modes agrees well with available literature,31 where co- and cross-polarization experiments were presented.Accordingto ref , bulk 1Tg modes should be constant, while the angular plots of the Eg modes should show a four-lobbed shape. Surprisingly, angular plotsobtained by mechanically rotating the sample in the cryostat by 35°are almost unaltered for both, Ag and Eg modes,as shown in 3 A1g is always rotatedby the same angle with respect to the excitation laser polarization.This polarization rotation can be inverted by switching the ferromagneticstate. This behavior has not been observed in case of Raman scatteringfrom other TMDCs in which polarization properties were analyzed basedon Raman tensors and group theory.29 Importantly, assuming point group Dd3 instead of Ci3, as suggested in ref  configurations, where σ+ (σ−)means right-handed (left-handed) circularly polarized light . As can be seen, the intensity of some lines show considerabledifferences, which is the signature of Raman optical activity.34 The lines at 74, 100, 134, 243, 248, and 298cm–1 are more intense in σ+/σ–configuration, while the lines at 265 and 283 cm–1 are more pronounced in σ–/σ+ configuration. Whenwe compare this data to the linear polarization angular plots  with a main axis closeto 90°, which can be easily influenced by more intense neighboringlines, and it is not clear whether their angular plot is rotated tothe left or right.36 found in semiconducting TMDCs (like MoS2) because theyrequire the inversion symmetry to be broken, which is not the case.The observed polarization rotations are well reproduced with anantisymmetric Raman tensor in the form37 for yttrium aluminum garnet and introduced in 338 and by Barron39 to explain ROA for molecularlight scattering. For the Raman tensor in 40 It is worthnoticing that the antisymmetric Raman effects induced by “chiralitydensity” were also observed by Raman scattering for the heavyFermion superconductor URu2Si2.41 In contrast to 1T-TiSe2, chirality has not beenobserved for pristine 1T-TaS2.43 However, recent reports show that chiral CDWs can be induced in1T-TaS2 by Ti doping43 and inthe case of the light-induced hidden state.33 Our results showing an insusceptibility of the polarization propertiesupon sample rotation and the observed Raman optical activity indicatethat the studied sample is chiral. In accordance with recent resultsshowing that the CDW stacking in 1T-TaS2 can be influencedeven by low-power incoherent white light illumination,17 we believe that in fact with Raman spectroscopywe do not probe a pure CCDW state, but a light-induced “hidden”state. As shown by Gerasimenko et al.33 intertwined chiral charge orders can arise in this state, whichmay give rise to the observed optical activity. Another explanationinvolves the experimental backscattering configuration. Because ofthis configuration, only phonons with specific momentum directionare prefered and detected. Our room-temperature polarization-resolvedmeasurements (see Supporting Information) show that Raman modes visible in the NCCDW phase can be linearlypolarized with the polarization direction neither parallel nor perpendicularto the incident light polarization direction. This behavior impliesthat the observed optical activity is related to the superlatticeformation and that some signs of it can be detected in the NCCDW phase.Antisymmetric Raman tensors were experimentally observed by Koningstein,keff = k0 ± G = 0,where G is a reciprocal lattice vector. However,due to the Brillouin zone reduction in the CCDW phase, we obtain twoadditional families of Γ points: the second group includes k0′ = {g1, g2, −g1, −g2, g2 – g1, g1 – g2} and the third group includes k0″ = {g1 + g2, 2g1 – g2, g1 – 2g2,– g1 – g2, g2 – 2g1, 2g2 – g1}. A corresponding schematic drawing is presented in A question that remains to be answered is why we observe threetypes of Raman modes? One type has a main axis of 0° and twomore types are rotated left and right, respectively. One possibleexplanation may be given by the superlattice reconstruction. For theundistorted lattice there is one Γ point of the first Brilloiunzone with an effective wavevector 2 showed that the angles of the main axis oflinearly polarized Raman modes cannot be explained in terms of semiclassicalmodels. We propose that they can be understood in terms of opticalactivity. Our results obtained with circularly polarized light indeedshow the expected Raman optical activity, which requires 1T-TaS2 in the CCDW phase to be chiral. The observed chirality canbe explained based on recent findings showing that a chiral CDW ordermay be present in the light-induced state of 1T-TaS2. Thisexplanation suggests that care must be taken when using laser-basedmethods to study CDW compounds, since light illumination may havean impact on the final properties. The polarization-resolved resultsare repeatable and do not depend on the position of the excitationspot on the sample, which proves that the observed effects are macroscopiccrystal properties and cannot be attributed to local microscopic defects.Our results show that full polarization-resolved Raman spectroscopy,in contrast to the commonly performed cross/co linear polarizationmeasurements, is a simple yet very powerful technique that allowsstudying the chirality of complex opaque materials on a macroscopicscale.Weshow that polarization-resolved Raman spectroscopy allows oneto study complex phenomena, especially in the case of opaque bulkmaterials. Polarization-resolved Raman spectroscopy measurements ofbulk 1T-TaS


Nicotiana benthamiana) compared to conventionally-designed gRNAs—reducing detectable viral RNA by >30-fold, despite lacking perfect complementarity with either of their targets and, when multiplexed, reducing viral RNA by >99.5%. Pairs of pgRNA-targetable sequences are abundant in the genomes of RNA viruses, and this work highlights the need for specific approaches to the challenges of targeting viruses in eukaryotes using CRISPR.CRISPR effector Cas13 recognizes and degrades RNA molecules that are complementary to its guide RNA (gRNA) and possesses potential as an antiviral biotechnology because it can degrade viral mRNA and RNA genomes. Because multiplexed targeting is a critical strategy to improve viral suppression, we developed a strategy to design of gRNAs where individual gRNAs have maximized activity at multiple viral targets, simultaneously, by exploiting the molecular biophysics of promiscuous target recognition by Cas13. These “polyvalent” gRNA sequences (“pgRNAs”) provide superior antiviral elimination across tissue/organ scales in a higher organism (  •Algorithm to design single gRNAs to target multiple viral sequences simultaneously•Pairs of targetable sequences are abundant in the genomes of RNA viruses•Validated in plants with Cas13, reducing viral RNA >30-fold versus conventional gRNAs•Approach also works in viral diagnostics and with other CRISPR effectors like Cas9 Biocomputational method; Computational bioinformatics; Biological sciences tools Class II CRISPR effectors  like Casin vitro  can provide superior antiviral elimination versus an infectious RNA virus compared to conventionally-designed gRNAs. We show that (1) despite lacking perfect complementarity to their targets, single pgRNAs with Cas13 targeting two viral sites can robustly suppress viral propagation in whole N. benthamiana plants across the organ/tissue scale, (2) that treatments with pgRNAs are better than those using a single perfectly-complementary “monovalent” gRNA—in some cases further reducing detectable viral RNA by an additional order of magnitude (30- to 40-fold) compared with “monovalent” gRNAs—and with fewer components single pgRNAs are at least as effective as and often better than treatments using two of their multiplexed “monovalent” gRNA counterparts; and (3) that treatments using multiplexed pgRNAs  perform even better, reducing detectable viral RNA in planta by >99.5%. in vitro, we show that, despite imperfect complementarity with their targets, pgRNAs can be engineered to trigger Cas13’s “collateral activity” for viral detection applications at viral target pairs with sequences diverging by up to 25% and that they can be designed to direct DNA-targeting CRISPR-Cas9 to degrade multiple DNA targets ex vivo with sequence divergence up to 40% (with mismatches at 8 out of 20 bp).Here, we present a computational algorithm to efficiently design pgRNA sequences optimized for activity at multiple viral targets, simultaneously, and experimentally demonstrate that using pgRNAs with Cas13 in a higher organism  with predicted activity at both their targeted sites ranking in the top quartile of all “monovalent” gRNAs for that virus and no significant homology/predicted activity versus the human transcriptome for 53 of the 59 human-hosted (+) ssRNA viruses or expressed viral mRNA sequences in the NCBI Reference Sequence database  (N. bethamiana) and infected N. benthamiana with a TMV replicon (TRBO-GFP) via Agrobacterium tumefaciens-mediated transformation into its leaves (A. tumefaciens-mediated transformation and T-DNAs to express either one or two multiplexed gRNAs or pgRNAs (N. benthamiana transcriptome by ensuring that each pgRNA contains at least 8 or 10 mismatches (<57–65% complementarity) with all sequenced N. benthamiana RNA transcripts  and again, could only find off-targets with >8 or even 10 mismatches for the 3 pgRNAs tested. We otherwise found no evidence of disruption of “off-target” cellular RNA levels when introducing the RfxCas13d and pgRNAs into the plants   or no gRNA C–2E. Whiin vivo in suppressing an active viral infection.The improved performance by the pgRNAs relative to their monovalent counterparts is remarkable considering that the pgRNA spacer sequence contains up to three or four imperfectly  complementary or mis-paired nucleotides with each of its two targets B. One exN. benthamiana through some as-yet-unknown mechanism, perhaps related to a similarly modest silencing effect previously reported in N. bethanamiana when gRNAs were expressed in the absence of any Cas enzymes  that were at least similar to and often better than those plants undergoing multiplexed expression of two of their (perfectly-complementary) “monovalent” counterparts . Multiplexed expression of two sets of the three pgRNAs—together targeting four viral targets simultaneously with two guides—further reduced viral RNA levels by an order of magnitude, in two cases to 0.33–0.6% viral RNA in the leaves compared to plants expressing the non-targeting gRNA. A third multiplexed pgRNA set “only” reduced viral RNA levels to 5%, levels equivalent to multiplexed monovalent gRNAs, which may have been a result of a predicted partial base pairing interaction between the two pgRNAs when both are expressed simultaneously . The ant enzymes . These rWe also note that in practice and particularly in non-transgenic organisms or cell lines, even generating arrays for multiplexed expression of gRNAs targeting four distinct sites can be non-trivially challenging for any CRISPR effector, including Cas13 . The abitrans cleavage” activity or “collateral activity” that occurs after target recognition and conformational change (Leptotrichia wadei (LwaCas13a), it has been found that multiplexed use of multiple gRNAs improves viral detection sensitivity  by the U.S. Food and Drug Administration, and purified LwaCas13a enzyme is also commercially available, but LwaCas13a has not been as exhaustively characterized as RfxCas13d with regards to tolerance to mismatches. We hypothesized that if the major contribution to activating Cas13 RNAse activity was the stability of base-pairing between gRNA spacer and target, we could use the mismatch sensitivities for RfxCas13d to provide at least a starting point or basis for designing pgRNAs for LwaCas13a for viral detection applications. This proved to be the case: despite SHERLOCK and the activation of collateral activity having been reported to be sensitive to single-nucleotide polymorphisms in their targets, , we founvergence D. These in vitro viral diagnostics, we generated a series of 23 pgRNAs with high predicted activity (using RfxCas13d design rules) at 15 target pairs found in SARS-CoV-2  implies their use compared to perfectly-matched gRNAs might increase assay robustness with regards to genetic variation across clinical variants, particularly under conditions where it might not be likely for variants to have so many mutations at both targeted sites simultaneously. For example, a variant with a large indel at one site might not be detectable in an CRISPR-based viral detection assay using a monovalent gRNA targeted to that site, but it is less likely that a variant will have large indels at all of the multiple sites recognized by a single pgRNA.These results suggest that pgRNAs are suitable for Additionally, the design principles of pgRNAs could also be applied to develop broad-spectrum Cas13-based viral diagnostics that are tolerant to polymorphisms across a viral family , e.g., fStreptococcus pyogenes (SpyCas9), which recognizes and introduces double-strand breaks into dsDNA targets  and measured the cleavage activity of purified Cas9 RNPs at those sites ex vivo  can exhibit stronger viral suppression despite mismatches with viral targets during infection of a higher organism relative even to perfectly-complementary “monovalent” targeting across organ and tissue scales, and (3) may in fact be optimal for applications of CRISPR antiviral diagnostics, prophylactics, and therapeutics. pgRNAs can also be synergistically integrated into strategies currently used in CRISPR antivirals to suppress mutational escape, such as gRNA multiplexing and targeting regions of high sequence conservation (or pairs of targets with high sequence conservation)   and to eWe emphasize that pgRNAs are engineered to target two specific viral sites, not to generally allow their CRISPR effector to be more prone to “off-target activity” or to tolerate more mismatches than any other gRNA or CRISPR effector using conventional gRNA designs. Both Cas13d and Cas9 have well-characterized natural tolerances to mismatches between their gRNA spacer sequence and target that we exploit for the specific targeting of two selected viral sites. For example, as mentioned above, Cas13d requires at least 18 nts of complementarity (fewer than 5 mismatches for a 23 nt spacer) to its target for stable binding and target cleavage to occur . In Figuin planta, it appears the “polyvalency” of pgRNAs can compensate for any potential reduction of activity at any of its target sites in a way that improves their antiviral activity with Cas13 over their perfectly-complementary monovalent counterparts.It is remarkable and perhaps counterintuitive that despite multiple mismatches and/or non-canonical base pairings with any of their viral targets, pgRNAs with Cas13 could significantly outperform their monovalent counterparts that have perfectly complementary to viral sequences—in one case by decreasing viral RNA across a tissue by 30- to 40--fold relative to the monovalent gRNAs. Single pgRNas were also shown they can outperform their monovalent gRNAs even when those pairs of monovalent gRNAs were multiplexed. Though further investigation is necessary, we propose that this improvement of pgRNAs in ability to suppress viral propagation may be related to the kinetics of CRISPR effector recognition of a rapidly replicating RNA virus, a property that may be more important in antiviral applications than for other applications of CRISPR biotechnologies. That is, when performing multiplexed targeting with N distinct “monovalent” gRNAs, the amount of transfected CRISPR effector is likely limiting, and so this multiplexing can be thought to effectively reduce the number of effectors per target by 1/N. Although multiplexed targeting likely helps in preventing viral mutagenic escape, this effective reduction in effector concentration per target may have critical implications in the rate that each CRISPR effector can find and degrade their viral target on a rapidly replicating RNA virus in a eukaryotic cell. In contrast, a CRISPR effector with single pgRNA that can recognize N viral sites while still maintaining high activity at each site now has N-fold the number of viral targets it can potentially recognize, which would provide an enhancement in the rate of viral recognition per effector even over effectors with perfectly matched “monovalent” gRNAs that can only recognize a single viral site. It is perhaps this influence on the kinetics of effector recognition and degradation—which would potentially be less critical in gene editing applications or even in applications of Cas13-mediated “knock-down” of mRNA transcripts from a discrete set of chromosomal genes than for a viral target—that results in improved viral suppression. Based on our results N. benthamiana plants as a proof-of-concept to demonstrate the superiority of pgRNAs in antiviral applications of CRISPR, work in our laboratory is ongoing to determine whether pgRNAs can also be effective against human viruses in human cell lines. The prospect of CRISPR antiviral therapeutics is promising because they can be rapidly reconfigured in the face of emerging viral pathogens and could potentially be delivered as a transient antiviral agent during an acute infection  cell lines there is reason to believe pgRNAs should do so in animals as well. In addition, although polyvalent targeting of divergent was demonstrated eric.josephs@uncg.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Eric Josephs .Seeds for iversity . N. bentA. tumefaciens strain GV3101 were grown on LB -agar plates containing antibiotics appropriate for the plasmids propagated by the bacteria at 28°C. Single colonies were grown overnight at 28°C in LB containing antibiotics appropriate for the plasmids propagated by the bacteria prior to agro-infiltration of plants.E. coli strain TOP10 (Thermo Fisher) were grown on LB-agar plates containing antibiotics appropriate for the plasmids propagated by the bacteria at 30 or 37°C. Single colonies were grown overnight at 30°C or 37°C in LB media containing antibiotics appropriate for the plasmids propagated by the bacteria.https://github.com/ejosephslab/pgrna (https://doi.org/10.5281/zenodo.7126522). Viral genome sequences used to identify targets are listed in Step 1: Estimate activity at different targets (‘protospacers’). “On-target” activity for every potential target in a viral genome is found using sgRNA Designer for Cas9  or cas13Step 2: Identification of Targetable Pairs with high homology. Every potential target is aligned to every other potential target, and pairs with >70% sequence identity (≥14 nt identity for 20 nt Cas9 targets and ≥16 nt identity for 23 nt Cas13d targets) are identified.i.e. 4n candidates for target pairs with n differences between sequence. A mismatch penalty  between the candidates and each of the target pairs was calculated using the multiplicative approach  at each of the sites of sequence divergence between the target pairs, ch i.e., C right. ch i.e.,  If the sN. benthamiana we used transcriptome assembly v5  with the Bioinformatics toolbox installed or Python 3.1 with the biopython package  using coEstimates of the predicted CRISPR activity at sites not perfectly targeted by the gRNA/pgRNA spacer sequence were generated by calculating the Cutting Frequency Determination (CFD) score , 2016. TAll complete sequences of all RNA viruses with human, mammal, arthropoda, aves, and higher plant hosts found in the NCBI Reference Sequence database were subjected to a brute force direct  alignment for each of their 23 nt sequence targets to each other, considering only sequence polymorphisms at the same site. We considered only the (+) strand, as even for (−) and dsRNA viruses these sequences would match the vast majority of mRNA sequences. Only targets lacking polynucleotide repeats  were considered viable targets. Targets derived from different segments or cDNAs of the same viral strain were considered together. In total: arthropoda , aves (111), mammal (496), higher plant/embrophyta (691), and human (89)-hosted viruses were considered  screenedR. flavefaciens (RfxCas13d) flanked by two nuclear localization signal (NLS) was amplified from plasmid pXR001 (Addgene #109049) using Q5 high fidelity of DNA polymerase (NEB). Similarly, overlap extension PCR was performed to amplify plant expression vector pB_35S/mEGFP (Addgene #135320) with ends that matched the ends of the Cas13 product so RfxCas13d expression would be under the control of 35S Cauliflower mosaic virus promoter. The PCR products were treated with DpnI (NEB), assembled together in a HiFi DNA assembly reaction (NEB), transformed into NEB10b cells (NEB), and grown overnight on antibiotic selection to create plasmid pB_35S/RfxCas13. Successful clones were identified and confirmed by sequencing followed by transformation into electro-competent A. tumefaciens strain GV3101 (pMP90).The DNA sequences of the plant codon optimized Cas13d-EGFP with the Cas13d from A. tumefaciens strain GV3101. Multiplexed expression of two crRNAs was achieved by ligating  oligos for two individual crRNAs (hairpin + spacer) together with an internal 4 nt “sticky-end” and into SPDK3876 so both crRNAs would be expressed on a single transcript.Single stranded oligonucleotides corresponding to “monovalent”, non-targeting (NT), and “polyvalent” gRNAs were purchased from Integrated DNA Technologies , phosphorylated, annealed, and ligated into binary vector SPDK3876 (Addgene #149275) that had been digested with restriction enzymes XbaI and XhoI (NEB) to be expressed under the pea early browning virus promoter (pEBV). The binary vector containing the right constructs were identified, sequenced and finally transformed into A. tumefaciens strain GV3101. Single colonies were grown overnight at 28 degrees in LB media . The overnight cultures were then centrifuged and re-suspended in infiltration media  and incubated to 3–4 hours at 28 degrees. The above cultures were mixed to a final OD600 of 0.5 for CasRX-NLS-GFP-pB35, 0.1 for PLY192 (TRV RNA1), 0.1 for RNA2- crRNAs and 0.005 for TRBO-GFP and injected into healthy leaves of five to six-week-old N. benthamiana plants grown under long-day conditions . A total of four leaves for each gRNA were infiltrated. Three days after transfection, leaves were cut out and photographed under a handheld UV light in the dark, and stored at −80°C before subsequent analysis.In addition to pB_35S/RfxCas13 and the SPDK3876’s harboring gRNA sequences (TRV RNA2), PLY192 (TRV RNA1) (Addgene #148968) and RNA viruses TRBO-GFP (Addgene # 800083) were individually electroporated into TM SYBR Green pre-formulated 2x master mix (Applied Biosystems). Relative expression levels based on fold changes were calculated using the ddCT method. Cycle 3 GFP mRNA expression levels from the TRBO-GFP replicon were normalized against transcripts of the tobacco PP2A, then to compare gRNAs across experiments, mRNA expression levels within a batch of plants are normalized relative to the mean GFP levels in experiments with non-viral targeting controls  and the yield was quantified using a nanodrop. A total of 1 μg RNA from control (NT gRNAs) and experimental samples were used for DNase I treatment  followed by reverse transcription using a poly-dT primer and the Superscript III First Strand cDNA Synthesis System for RT–PCR (Invitrogen). Quantitative PCR was performed on Quant studio 3 Real-Time PCR System from Applied Biosystems using iTaq PowerUPCD46 transcript sequences (Leptotrichia wadei Cas13a (LwaCas13a) enzyme  was preincubated with each pre-synthesized gRNA (CD46 targets)  or in vitro transcribed gRNA (SARS-CoV-2 targets)  in a total volume of 5 μL for 10 min at room temperature, followed by the addition of 16 μL of synthetic dsDNA template  at varying concentrations . A master mix containing 0.5 μL of T7 RNA polymerase , 1 μL of 25 mM rNTPS , 0.23 μL 1M MgCl2 , 0.5 ul HEPES , 0.63 μL of RNAseH inhibitor (NEB), 1.56 μL RNAse Alert Reporter (IDT), and 0.58 ul of nuclease-free water (Invitrogen) were assembled on ice and 4 μL added to the mixture containing the DNA template and preincubated Cas13 RNP. 25 μL of each preassembled reaction was added to a 384-well plate (Black/Clear Bottom) and loaded into a preheated fluorescence microplate reader (Promega GloMax Explorer) at 37°C. Data readouts were collected every 5 min for 1hrat an excitation peak at 480 nm and an emission peak at 520 nm. List of all (p)gRNAs screened is found in Initial screens were performed using synthetic dsDNA (∼300 bp) containing a T7 promoter located upstream of a specific target sequence derived from either SARS-CoV-2 C and S7 equences B in two Specificity of Cas13 collateral activity was evaluated using dsDNA fragments that were not complementary to the gRNAs being tested to confirm that activation of collateral activity as well as human universal RNA (10 tissues) , and total human lung RNA , was also used at 1 and 3 ug, respectively per reaction.Heat-inactivated SARS-CoV-2 RNA from respiratory specimens, deposited by the Centers for Disease Control and Prevention, was obtained through BEI Resources, NIAID, NIH: Genomic RNA from SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52285 (American Type Culture Collection (ATCC) VR-1986HK). In a SHERLOCK-type reaction, 1 μL of heat-denatured SARS-CoV-2  was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) with 3.4 μL of primer (0.5 μM) in a final volume of 16 μL and PCR-amplified by the addition of 2 μL of reverse and forward target primers (2 μM) and 20 μL of 2 x OneTaq Master Mix (NEB) in a final volume of 40 μL under standard thermocycler conditions . Reactions were diluted to 4, 40, or 400 cp/uL of the starting viral material  for eachex vivo validation of the pgRNA design protocol, or PCR amplification of targets in plasmids using primers listed in Cas9 DNA targets in Plasmids were purified using Monarch Plasmid Miniprep Kit following standard protocols . PCR amplification was carried out using 4.5 ng of plasmid DNA, downstream and upstream PCR primers  at a final concentration of 0.2uM, and Taq 2x Master Mix  following standard thermocycling protocols. Amplified PCR targets were purified using a Monarch PCR and DNA cleanup kit (NEB) following standard protocols. DNA oligonucleotides were hybridized to form duplex DNA targets by using equal molar concentration of oligos  to a final concentration of 10uM in nuclease-free IDT Duplex buffer. Reactions were heated to 95°C for 2 min and allowed to cool to room temperature prior to the reaction assembly.Single guide RNA (sgRNA) was synthesized by using the EnGen sgRNA synthesis Kit  following standard protocols. DNA oligos (IDT) were designed to contain a T7 promoter sequence upstream of the target sequences with an initiating 5′- d(G), as well as overlapping tracrRNA DNA sequence at the 3′ end of the target. The sgRNA was purified using Monarch RNA Cleanup Kit (NEB) and quantitated using standard protocols.ex vivo validation of the pgRNA design protocol to a universal synthetic tracer RNA oligo (IDT). To hybridize oligos, equal molar concentration of oligos were combined in IDT duplex buffer to a final concentration of 10uM. Reactions were heated to 95°C for 2 min and allowed to cool to room temperature prior to the reaction assembly.Duplex CRISPR gRNAs (cRNA:tracrRNA) was generated by hybridizing synthetic RNA oligos listed in S. pyogenes (NEB) was diluted in 1x NEB Buffer 3.1. prior to the reaction assembly. Cas9 cleavage activity was performed using either PCR-amplified targets, whole plasmid, or hybridized DNA oligos containing desired targets using standard methods. Briefly, Cas9 was preincubated with either a sgRNA or duplex gRNA (crNA:tracRNA) for 5 min at equal molar concentrations in 1x NEB Buffer 3.1 (NEB) in a volume total of 10 ul. Reactions were incubated for 5–10 min at room temperature. Target DNA was then added to the reactions, NEB Buffer 3.1 was added back to a final concentration of 1x, and nuclease-free water was added bringing the final volume to 20 ul. The final reaction contained 100 nM Cas9-CRISPR complex and 10 nM of target DNA. Similar reactions without the addition of gRNAs to Cas9 were used as a control for uncut DNA. Reactions were incubated at 37°C for 1 h, followed by the addition of 1 unit of Proteinase K and further incubation at 56°C for 15 min. Reactions were stopped by the addition of one volume of purple Gel Loading dye (NEB).Cas9 Nuclease from Fragments were separated and analyzed using a 1.5% Agarose gel in 1xTAE and 1X SYBR Green 1 Nucleic Acid Gel Stain , and fluorescence was photographed and measured . Cas9 cleavage efficiency was calculated from fluorescence intensity at each band.All complete SARS-CoV-2 genomic sequences available from the NCBI Virus database were downloaded on November 23, 2020 . For each of the 205 target pairs possessing biophysically feasible pgRNA candidates, we aligned (no gaps) each target sequence to each genome to determine the closest matching sequence. Alignments containing ambiguous nucleotide calls were not included. Sequence variants were grouped together, with a minimum prevalence of 0.1%, with the fraction of hits by the most prevalent group being considered the sequence conservation reported.Data was quantified using the methods described above, and the statistical analysis was performed using Microsoft Excel and MATLAB . The numbers of samples (N) are plotted explicitly in each graph and/or recorded explicitly in the supplementary table where the analysis is performed. Hypothesis testing regarding differences in the mean quantities of viral RNA were performed in MATLAB using two-sided t-tests.


However, studies on the contribution of this antiporter to ischemic brain damage in vivo are more limited. Since embolic or thrombotic transient or permanent occlusion of a cerebral blood vessel eventually leads to brain infarction in most stroke cases, we evaluated the contribution of Sxc– to cerebral ischemic damage by comparing brain infarction between mice naturally null for SLC7a11 (SLC7a11sut/sut mice) – the gene the encodes for the substrate specific light chain for system xc– – with their wild type (SLC7a11 + / +) littermates following photothrombotic ischemic stroke of the middle cerebral artery (PTI) and permanent middle cerebral artery occlusion (pMCAo) rendered by cauterization. In the PTI model, we found a time-dependent reduction in cerebral blood flow that reached 50% from baseline in both genotypes 47–48 h post-illumination. Despite this, a remarkable reduction in incidence and total infarct volume of SLC7a11sut/sut mice was revealed 48 h following PTI as compared to SLC7a11+/+ mice. No difference in injury markers and/or infarct volume was measured between genotypes when occlusion of the MCA was permanent, however. Present data demonstrate a model-dependent differential role for Sxc– in focal cerebral ischemic damage, further highlighting that ischemic severity activates heterogeneous biochemical events that lead to damage engendered by stroke.Understanding the mechanisms underlying ischemic brain injury is of importance to the goal of devising novel therapeutics for protection and/or recovery. Previous work in our laboratory and in others has shown that activation of cystine/glutamate antiporter, system x Surrounnditions , 2012. Oectively , as wellectively . In vivoas found  along wias found . All togSlc7a11, the gene that encodes for the substrate-specific light-chain for Sxc– (xCT) (+/+) or lacking system xc– (Slc7a11sut/sut)—were derived from F1 heterozygous (Slc7a11+/sut) breeding units created by crossing Slc7a11sut/sut C3H/HeSnJ male mice [Jackson Laboratories (JAX) Stock #001310] with Slc7a11+/+ C3H/HeSnJ female mice . Mice were only used up to the F3 generation.This study was conducted in accordance with the National Institute of Health guidelines for the use and care of experimental animals as approved by the Institutional Animal Care and Use Committee. We utilized male mice on the C3H/HeSnJ background that have a naturally occurring deletion mutation in c– (xCT) . Experimvia PCR analysis of tail genomic DNA samples as described  were physically held so that they would become accustomed to being touched. On the surgical day, mice were brought into the procedure room, weighed, and allowed to acclimatize for at least 1 h. Investigator was blind to mouse’s genotype at time of experimentation  and during all subsequent analyses. All mice were sacrificed 48 h after the induction of cerebral ischemia as described below. via photothrombosis via laser irradiation of the photosensitive compound Rose bengal (®). Freshly made Rose bengal  was then injected retro-orbitally  using a 28-gauge, 1/2 inch, 0.5 ml insulin needle and syringe. Three minutes after injection, the frame containing the mouse was mounted on an upright microscope platform and the exposed but otherwise intact skull was illuminated through the 1.5 mm diameter hole for 2 min using a 10x objective with a green light of bandwidth 540–580 nm by means of an X-cite 120Q light source to activate the dye to induce a thrombus. A heating pad was used to maintain mouse’s body temperature (36.5–37°C) during and following the procedure. Mortality with this procedure is very low with only one mouse of each genotype lost during surgery.Cerebral ischemic damage was induced e bengal . Mice, f3) and Ai = mean infarct area of each section derived from two measurements and D represents the distance between 2 sections (Ischemic damage was determined 48 h after surgery. Mice were perfused with cold PBS followed by 4% (v/v) paraformaldehyde (PFA). After perfusion, brains were removed and post-fixed in 4% PFA/PBS for 12 h and then placed in 20% (w/v) sucrose for an additional 12–24 h (4°C). Tissue was then embedded in Tissue Tec OCT and solidified in liquid nitrogen. Brain sections (20 μm) cut serially through the rostro-caudal extent of each brain (−2.0 to −3.6 relative to bregma)  were stained with 0.5% thionin as described in detail . Images sections .®), followed by the retro-orbital injection and laser activation of Rose Bengal as described above. After the activation, the metal sheet was gently removed, and mouse was immediately moved to the imager to access the real time blood flow for 30 sec (time zero). The incision was sealed lightly by glue, after which the mouse was put back into their cage containing a heating-pad and allowed to recover. Mice were re-anesthetized, the incision gently re-opened and additional blood flow measurements taken over the same region at 4–6, 24, and 47–48 h post-illumination. Data at each time point was normalized to baseline for each mouse and expressed as mean percentage ± S.D.Laser Speckle Contrast Analysis was used to measure microvascular cerebral blood flow in the right MCA territory where the Rose Bengal was activated  in a separate cohort of mice  for 30 min at 37°C in the dark. Slices were then carefully transferred into 10% formalin for 24 h. Images (300 dpi) were captured by scanning (Epson Perfection 3170). The non-injury areas of both contralateral and ipsilateral hemispheres—denoted by red staining—were measured using NIH ImageJ for all sections by two experimenters blind to the genotype of the mice and infarct area and volume determined. Infarct area (A) was determined by subtracting the non-injured ipsilateral area from the area of the contralateral hemisphere. Total infarct volume (mm3) was calculated using the following equation: V = Ai × 2 + Ai × 2 + Aii × 2 + Aiii x 2 + Aiv with V = total infarct volume and Ai = infarct area of each section with 2 representing the distance between slices   and cauterizing the right middle cerebral artery (MCA) distal to the striatal branch as described in ., 2 mm) .® blocking buffer; at 25°C for 1 h) and then probed  with an anti-α Fodrin monoclonal antibody  and a mouse monoclonal antibody directed against β-actin to correct for protein loading . Species-specific secondary antibodies labeled with spectrally distinct IRDye ® fluorescent dyes  were used to detect primary antibodies (1 h at 25°C). Results were recorded on LI-COR ODYSSEY ® Fc Imaging system (LI-COR Biosciences) and protein levels quantified using Image Studio 3.1 . Results were obtained by normalizing fodrin protein levels to their respective β-actin levels and expressed as mean fold change over the corresponding contralateral hemisphere (set to 1).To measure spectrin breakdown products, shown previously to correlate nicely with final infarct volume , contralvia two-tailed Mann Whitney U-test. Exact or adjusted p-values are included in text, in a table and/or described in figure legends as appropriate.All statistical analyses and graphics were compiled using GraphPad Prism . Cerebral blood flow (CBF) were analyzed using a mixed effects model with the Geisser-Greenhouse correction for normality followed by Šídák’s multiple comparisons test. αII-spectrin breakdown products (SBDPs) where analyzed using two-way ANOVA of log transformed data [y = log(y)] followed by Šídák’s multiple comparisons test. Infarct volumes were analyzed c– with overall infarct volume being significantly smaller  in SLC7a11sut/sut as compared to SLC7a11+/+ littermate controls (+/+ mice (12/13) had quantifiable cortical infarction as compared to just 25% (4/16) of SLC7a11sut/sut mice (p = 0.005). Differing amounts of occlusion could not account for the dissimilarity in PTI-mediated damage as similar decreases in rCBF starting at ≈25–30%  measured directly after laser illumination progressing to ≈50% reduction of pre-illumination levels  by the time of sacrifice occurred irrespective of the mouse’s genotype (p < 0.0001) with post hoc analyses revealing a significant reduction from baseline in SLC7a11+/+ mice occurring at 24 and 48 h, respectively. In contrast, significant changes from baseline occurred at all-time points post-illumination in SLC7a11sut/sut mice. Pertinently, no significant between genotype differences was found at any time post-illumination  (p = 0.925).In contrast to what we found with PTI, a well demarcated infarct was visible 48 hr following pMCAo in all mice regardless of genotype . Analysi> 0.999) . The levc– activity in rat brain in vivo following transient focal ischemia produced via intraluminal occlusion of MCA followed by reperfusion  but not severe (pMCAo) focal cerebral ischemia in mice.Occlusion of a major blood vessel, most notably the middle cerebral artery , leads terfusion . The modmination  at the tlization . PresentSlc7a11 gene  when subjected to cerebral ischemia followed by reperfusion  and late time points (1–3 days), indicating a role for Sxc– as a source of extracellular glutamate post-ischemia  or transiently .Studies in genetically modified mice are useful for potential target identification. However, in the interest of therapeutic development, it is important to note that in keeping with the observations described above, pharmacological inhibition of Sxammation  and infaammation  subsequeThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Syracuse University’s Institutional Animal Care and Use Committee.YH and SH: conceptualization, methodology, funding acquisition, writing of original draft, reviewing and editing, and statistical analysis. YH: investigation. SH: resources and supervision. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.