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

Abstract

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.

Optimized nickase- and nuclease-based prime editing in human and mouse cells

“…However, the efficiency was low, and installation of the PE mutations often induced large deletions due to concurrent dual nicking. Although PE2 reduced indel generation, on-target editing efficiency was also decreased ( 10 , 12 ). Given the relatively high efficiency of PE-nuclease in cell culture experiments, we sought to assess PE-nuclease efficiency in microinjected zygotes.…”

Expansion of the prime editing modality with Cas9 from Francisella novicida

“…Cells were subcultured every 48 h to maintain 70% confluency. For target sequence editing, 2 × 10 5 HEK293FT or HeLa cells were transfected with plasmids expressing pegRNAs (240 pmol), the prime editor expression plasmid [SpCas9(H840A)-RT (Addgene no. 132775), FnCas9(H969A)-RT (developed in this study)], and nicking sgRNA (60 pmol) via electroporation using an Amaxa electroporation kit (V4XC-2032; program: CM-130 for HEK293FT cells, CN-114 for HeLa cells).…”

Prime editing – an update on the field

“…Recently Gao et al compared HDR and PE2 to edit the CArG box transcription factor binding site in mice [ 14 ]. Analysis of founder mice revealed approximately twice as many successfully edited animals with HDR (20/37) versus PE2 (12/47).…”