Biased genome editing using the local accumulation of DSB repair molecules system

Nakade, Shota; Mochida, Keiichi; Kunii, Atsushi; Nakamae, Kazuki; Aida, Tomomi; Tanaka, Kohichi; Sakamoto, Naoaki; Sakuma, Tetsushi; Yamamoto, Takashi

doi:10.1038/s41467-018-05773-6

Cited by 23 publications

(25 citation statements)

References 36 publications

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“…1B). Consistent with previous reports 13,22,23 , among the Cas9 variants, Cas9 WT -CtIP performed with a roughly 2-fold improvement in knockin efficiency compared to Cas9 WT , regardless of donor variant (Fig. 1D, 1G).…”

Section: G H Isupporting

confidence: 92%

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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Section: G H Isupporting

confidence: 92%

Cas9 fusions for precisionin vivoediting

Richardson

Steyert

Inen

et al. 2020

Preprint

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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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“…However, using this method we could not suppress mosaic mutations completely. Therefore, other approaches such as using exonucleases that have higher activity in porcine embryos and using a system that accumulates Trex2 at double-strand break sites [42] may reduce mosaic mutations further. Three replicate trials were carried out.…”

Section: Discussionmentioning

confidence: 99%

Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation

Yamashita

Kogasaka

Hiradate

et al. 2020

Journal of Reproduction and Development

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Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 μm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 μm) as compared to those from the control group (178.9 ± 9.0 μm). These results revealed that mTrex2 did not affect the development of preimplantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.

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Biased genome editing using the local accumulation of DSB repair molecules system

Cited by 23 publications

References 36 publications

Cas9 fusions for precisionin vivoediting

Cas9 fusions for precisionin vivoediting

Prime editing primarily induces undesired outcomes in mice

Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation

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