Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.

Genome biology 2021 Mar 16

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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. 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. 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.

Citation

Pan Gao, Qing Lyu, Amr R Ghanam, Cicera R Lazzarotto, Gregory A Newby, Wei Zhang, Mihyun Choi, Orazio J Slivano, Kevin Holden, John A Walker, Anastasia P Kadina, Rob J Munroe, Christian M Abratte, John C Schimenti, David R Liu, Shengdar Q Tsai, Xiaochun Long, Joseph M Miano. Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression. Genome biology. 2021 Mar 16;22(1):83