Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.
BACKGROUND
Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter.


RESULTS
Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders.


CONCLUSIONS
PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
['Gao|Pan|P|', 'Lyu|Qing|Q|', 'Ghanam|Amr R|AR|', 'Lazzarotto|Cicera R|CR|', 'Newby|Gregory A|GA|', 'Zhang|Wei|W|', 'Choi|Mihyun|M|', 'Slivano|Orazio J|OJ|', 'Holden|Kevin|K|', 'Walker|John A|JA|', 'Kadina|Anastasia P|AP|', 'Munroe|Rob J|RJ|', 'Abratte|Christian M|CM|', 'Schimenti|John C|JC|', 'Liu|David R|DR|', 'Tsai|Shengdar Q|SQ|', 'Long|Xiaochun|X|', 'Miano|Joseph M|JM|0000-0001-7522-3207']
Prime editing Gene expression Transcription Genome editing Mouse CRISPR