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.
Highlights
Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus
Here we show that the MMR pathway counteracts PE efficiency and fidelity, across different human immortalised and induced pluripotent stem cell lines, genomic loci and edit types
Activation of MMR was strictly dependent on the presence of two factors: a mismatch and a pre-existing nick in one strand that was less than 1 kb distant
Summary
Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. Following the targeting of a site-specific DNA double-strand break (DSB), the endogenous DNA end-joining pathways frequently repair this lesion in an error prone manner, leading to insertions or deletions (indels) that give rise to loss-offunction alleles[6]. This approach was further adapted to include either a single-stranded or double-stranded donor template containing the desired edit. The DSB is processed by homology-directed repair (HDR), which catalyses the insertion of the donor template that includes the edit Unlike the former approach, which generates random indels, the latter method permits the introduction of desired indels, as well as point mutations, into the genome[3]. Since HDR is inefficient, depends on potentially deleterious DSBs and requires cell division, alternative approaches were needed
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
