Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks

Abstract

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized.

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

“…2c, Supplementary Figs. [14][15][16]. Encouragingly, dC9-ABE8 variants only have a median 14% reduction in on-target DNA editing efficiencies relative to nickase-active ABE8s.…”

“…To date, seventh generation ABEs (ABE7) have enabled efficient A•T to G•C conversion in the genomes of humans 5 , mice [6][7][8] , bacteria 1 , plants 9,10 , and a variety of other species, reviewed here 11 . Many therapeutic targets, however, may benefit from a more active ABE with a broader editing window or improved compatibility with non-NGG nCas9s [12][13][14] as well as increased editing efficiencies in human cell lines 15 or when used in vivo 8 . The need for a more active version of ABE7.10 is the greatest when target adenines are positioned on the outer edges of the canonical ABE editing window (positions 3, 4, 7 and 8).…”

Strategies for the Treatment of Parkinson’s Disease: Beyond Dopamine

“…Active research efforts are currently underway to overcome these limitations. Recently one of the latest advances in CRISP technology was reported by Rees et al (2019) from Harvard University (Anzalone et al, 2019). In this new approach, Cas9 hybridizes to the target DNA site using a guide engineered RNA containing a complementary spacer.…”

Prime editing primarily induces undesired outcomes in mice

“…CRISPR/Cas-based approaches for targeted genome modification have revolutionized modern biology and hold great promise for therapeutic interventions for debilitating genetic disorders. In particular, engineered enzymes have given the gene editing field an everexpanding set of tools with increased fidelity (Kleinstiver et al, 2016;Slaymaker et al, 2016), altered target specificities (Chatterjee et al, 2018;Hu et al, 2018;Nishimasu et al, 2018;Walton et al, 2020), the ability to directly introduce specific changes to a target genome (Gaudelli et al, 2017;Komor et al, 2016;Nishida et al, 2016), or improved genome modification capabilities (Aida et al, 2015;Charpentier et al, 2018;Gu et al, 2018;Jayavaradhan et al, 2019;Nakade et al, 2018;Rees et al, 2019). However, these options continue to suffer from low efficiencies, high indel rates, a narrow scope of editing outcomes, and/or a reliance upon restricted sets of suitable protospacer adjacent motifs (PAMs) in close proximity to the target site.…”