Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair

Abstract

Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, the efficiency of PE varies widely and depends not only on the genomic region targeted, but also on the genetic background of the edited cell. Here, to determine which cellular factors affect PE efficiency, we carry out a focused genetic screen targeting 32 DNA repair factors, spanning all reported repair pathways. We show that, depending on cell line and type of edit, ablation of mismatch repair (MMR) affords a 2–17 fold increase in PE efficiency, across several human cell lines, types of edits and genomic loci. The accumulation of the key MMR factors MLH1 and MSH2 at PE sites argues for direct involvement of MMR in PE control. Our results shed new light on the mechanism of PE and suggest how its efficiency might be optimised.

Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites

“…PE do not require DSB intermediates for their editing activity, raising the possibility that L1 insertions might not occur frequently in their editing sites. Since HEK293T cells have been widely used to validate PE efficiency 64 , 65 , we used this cell line to assay for de novo L1-ORFeus insertions at editing sites targeted by various prime editors: PE2, PE3, and PE3b, as well as nick only for PE3 served as a nickase-Cas9 generated single-strand break (SSB) control, or nick only for PE3b served as a negative control since this nicking sgRNA binds only to the edited sequence 64 (Fig. 7a ).…”

Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants

“…The efficiency of genome editing varies with the target locus, distance to the protospacer adjacent motif (PAM) and mismatch repair proficiency of the cell model ( Chen et al, 2021 ; Ferreira da Silva et al, 2022 ). For example, the editing efficiency of CRISPR/Cas9 falls as distance from the PAM increases and stem cells show relative resistance to transfection compared to immortalized cell lines ( Madsen and Semple, 2019 ).…”

Peptide fusion improves prime editing efficiency

“…In summary, through screening 12,000 peptide-PE2 fusion proteins using PepSEq, a sensitive, NGS-based self-editing platform, we identify a prime editor that consistently increases editing efficiency across dozens of targets in four human and mouse cell lines. There have been a number of orthogonal approaches that have recently been shown to increase prime editing efficiency 5 , 14 , 17 – 19 . Prime editing has been shown to be inhibited by the MMR pathway and improved by co-expression of a dominant negative MLH1 isoform 14 , 17 .…”

“…There have been a number of orthogonal approaches that have recently been shown to increase prime editing efficiency 5 , 14 , 17 – 19 . Prime editing has been shown to be inhibited by the MMR pathway and improved by co-expression of a dominant negative MLH1 isoform 14 , 17 . However, this improvement is only observed for certain types of edits, presumably due to differential recognition by distinct MMR subcomplexes.…”