Precise genomic deletions using paired prime editing

Abstract

Technologies that precisely delete genomic sequences in a programmed fashion can be used to study function as well as potentially for gene therapy. The leading contemporary method for programmed deletion uses CRISPR/Cas9 and pairs of guide RNAs (gRNAs) to generate two nearby double-strand breaks, which is often followed by deletion of the intervening sequence during DNA repair. However, this approach can be inefficient and imprecise, with errors including small indels at the two target sites as well as unintended large deletions and more complex rearrangements. Here we describe a prime editing-based method that we term PRIME-Del, which induces a deletion using a pair of prime editing gRNAs (pegRNAs) that target opposite DNA strands, effectively programming not only the sites that are nicked but also the outcome of the repair. We demonstrate that PRIME-Del achieves markedly higher precision in programming deletions than CRISPR/Cas9 and gRNA pairs. We also show that PRIME-Del can be used to couple genomic deletions with short insertions, enabling deletions whose junctions do not fall at protospacer-adjacent motif (PAM) sites. Finally, we demonstrate that lengthening the time window of expression of prime editing components can substantially enhance efficiency without compromising precision. We anticipate that PRIME-Del will be broadly useful in enabling precise, flexible programming of genomic deletions, including in-frame deletions, as well as for epitope tagging and potentially for programming rearrangements.

A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening

“…40 Because prime editing with the PE2 approach produces primarily the intended edit or unedited sequence at the targeted site, neither of which represents an unwanted "endpoint", stable expression of editing components should result in accumulation of precise edits over time (Figure 1E). 17,[27][28][29][30][31]41,42 Results from our experiments with the HEK3 +1 T>A and DNMT1 +6 G>C edits confirmed this expectation, demonstrating continuous accumulation of intended edits over one month (Figure 1C) with minimal observation of unwanted byproducts or "errors" at either site (Figures 1D and S1B). Efficiencies of both substitutions were highest in cells with the optimized prime editor (PEmax) and when using an epegRNA in the absence of MMR (Figure 1C).…”

Theoretical Guarantees for Phylogeny Inference from Single-Cell Lineage Tracing

“…Recent strategies -such as pairing terminal deoxynucleotidyl transferase (TdT) with Cas9 [16] -have shown potential to increase indel diversity. Further, prime editors offer an avenue to more finely control the state distribution by dictating a-priori which indel will result in a given edit, though this technology has yet to be adopted for lineage tracing [41,42,43]. Fortunately, current CRISPR-Cas9 lineage tracing systems are capable of generating "un-problematic indel distributions".…”

Programming large target genomic deletion and concurrent insertion via a prime editing-based method: PEDAR

“…3h). Importantly, we show that PEDAR is able to introduce >10-kb target deletions and up to 60-bp insertions in cells, both of which are larger than what primer editors can generate 17,36 . Moreover, PEDAR can program target deletioninsertion editing in quiescent hepatocytes in mouse liver, where HDR is not favorable 37 .…”

“…When delivered with paired pegRNAs, PE-Cas9 couples target deletion and insertion to direct the desired genome edits. Our PEDAR system is similar to a recently developed paired prime editing method, called PRIME-Del, that can introduce 20-to 700-bp target deletions and up to 30-bp insertions 36 . Compared to PRIME-Del, PEDAR seems to be more error-prone, introducing higher fractions of direct deletion and imperfect deletion-insertion (Supplementary Fig.…”