CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites

Jayavaradhan, Rajeswari; Pillis, Devin M.; Goodman, Michael; Zhang, Fan; Zhang, Yue; Andreassen, Paul R.; Malik, Punam

doi:10.1038/s41467-019-10735-7

Cited by 135 publications

(128 citation statements)

References 51 publications

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“…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.…”

Section: Introductionmentioning

confidence: 99%

Prime editing primarily induces undesired outcomes in mice

Aida

Wilde

Yang

et al. 2020

Preprint

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SummaryGenome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

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Section: Introductionmentioning

confidence: 99%

Prime editing primarily induces undesired outcomes in mice

Aida

Wilde

Yang

et al. 2020

Preprint

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“…Furthermore, we demonstrated that this method can be used in combination with the CRISPR/Cas9 method to improve the efficiency of gene targeting. To enhance HDR, key factors involved in NHEJ, such as DNA-PKcs, Ku70, and DNA ligase IV, have been inhibited by widely used methods, such as shRNA-treatment, proteolytic degradation, or treatment with small molecules [38][39][40] . In HDR, the 5′-end of a DSB is resected to create ssDNA carrying a 3′ overhang.…”

Section: Discussionmentioning

confidence: 99%

“…However, HDR efficiency is limited by the presence of a competing repair pathway that is far more efficient, NHEJ. To enhance the frequency of HDR, global inhibition of key NHEJ factors has been the most widely studied strategy [38][39][40] . Although these approaches are effective in increasing the frequency of HDR, further improvements are required to achieve more practical HDR-based genome engineering.…”

Section: Direct Gene Targeting In Fertilized Mouse Eggs By Pronuclearmentioning

confidence: 99%

Enhanced homologous recombination by the modulation of targeting vector ends

Hirotsune¹,

Kanazawa²,

Jin³

et al. 2020

Sci Rep

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The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (cRiSpR) and cRiSpR-associated protein (cRiSpR/cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3′ overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHot), is a new and powerful method for genome engineering. The generation of targeted genome-modified animals provides an important approach for investigating gene functions in pathogenesis and gene therapy in humans. With conventional gene-editing methods, mutations are introduced through homology-directed repair (HDR) in embryonic stem (ES) cells. Usually, chimeric animals are generated by the injection of gene-targeted ES cells into wild-type (WT) blastocysts. Subsequent mating of chimeric animals produces mice carrying the targeted gene modification 1. Alternative methods exist in which DNA or mRNA that encode site-specific zinc finger nucleases (ZNFs) or transcription activator-like effector nucleases (TALENs) are directly injected into a fertilized egg to introduce DNA double-strand breaks (DSBs) at a specified locus, and these methods have been developed in a variety of species to target specific genomic sites 2-6. Recently, type II bacterial clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system-based gene-targeting tools have been applied for multiplex genome editing 7-9. Indeed, the CRISPR/Cas system has been demonstrated to drive both nonhomologous end joining (NHEJ)-based gene disruption and HDR-based precise gene editing 10,11. Furthermore, the CRISPR/Cas system is currently being applied to inducible multiplex gene regulation, genome-wide screens and cell fate engineering 12. Although the CRISPR/Cas system is an excellent method for genome modification, there are still limits to its application, including off-target effects and low gene replacement efficiency. HDR and classical nonhomologous end joining (C-NHEJ) are central cellular pathways involved in the repair of DSBs. In addition to these central pathways, other error-prone repair systems, known as alternative nonhomologous end joining (A-NHEJ) and microhomology-mediated end joining (MMEJ), are genetically independent from C-NHEJ. In C-NHEJ, the Ku heterodimer binds to DSB ends to protect DNA from extensive resection and unwinding. In MMEJ, DSB ends are resected, and the exposed DNA fragments that exhibit

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“…For our study, repair molecules were chosen based on previous reporters highlighting their impact on DNA repair outcomes 28,32,33,34,23,27 . We found that N-terminal fusion of Rad52 and eRad18 improved the knockin precision of Cas9.…”

Section: Discussionmentioning

confidence: 99%

“…Several groups have independently demonstrated that modulation of DNA repair pathways is an effective way to improve knockin efficacy and precision 26,13,23,27,28 . To further improve on the in vivo results using CtIP-fused Cas9, we returned to the in vitro BFP-to-GFP conversion assay with an HMEJ donor, and evaluated the impact of five DNA repair proteins (dn53BP1, TIP60, RNF169, Rad52, eRad18) on editing efficiency and precision when fused N-terminally to Cas9 or Cas9-CtIP (Fig.…”

Section: Compound Cas9-ctip Fusions With Erad18 and Rad52 Improve Edimentioning

confidence: 99%

Cas9 fusions for precisionin vivoediting

Richardson

Steyert

Inen

et al. 2020

Preprint

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Current Cas9 reagents can target genomic loci with high specificity. However, when used for knockin, on-target outcomes are inherently imprecise, often leading to unintended knockout rather than intended edits. This restricts applications of genome editing to ex vivo approaches, where clonal selection is possible. Here we describe a workflow using iterative high-throughput in vitro and high-yield in vivo assays to evaluate and compare the performance of CRISPR knockin reagents for both editing efficiency and precision. We tested combinations of Cas9 and DNA donor template variants and determined that Cas9-CtIP with in situ linearized donors display fold-increases of editing precision in cell lines and in vivo in the mouse brain. Iterating this process, we generated novel compound fusions, including eRad18-Cas9-CtIP that showed further fold-increases in performance. Continued development of precision editing reagents with this platform holds promise for direct in vivo knockin across model organisms and future targeted gene therapies.

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CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites

Cited by 135 publications

References 51 publications

Prime editing primarily induces undesired outcomes in mice

Prime editing primarily induces undesired outcomes in mice

Enhanced homologous recombination by the modulation of targeting vector ends

Cas9 fusions for precisionin vivoediting

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