RT Journal Article
SR Electronic
T1 Precise genomic deletions using paired prime editing
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.12.30.424891
DO 10.1101/2020.12.30.424891
A1 Junhong Choi
A1 Wei Chen
A1 Chase C. Suiter
A1 Choli Lee
A1 Florence M. Chardon
A1 Wei Yang
A1 Anh Leith
A1 Riza M. Daza
A1 Beth Martin
A1 Jay Shendure
YR 2021
UL http://biorxiv.org/content/early/2021/01/01/2020.12.30.424891.abstract
AB 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.Competing Interest StatementThe University of Washington has filed a patent application based on this work, in which J.C., W.C., and J.S. are listed as inventors.