2021
DOI: 10.1089/crispr.2020.0034
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A Novel Set of Cas9 Fusion Proteins to Stimulate Homologous Recombination: Cas9-HRs

Abstract: CRISPR-Cas9 has revolutionized genetic engineering. However, the inability to control double-strand break (DSB) repair has severely limited both therapeutic and academic applications. Many attempts have been made to control DSB repair choice. However, particularly in the case of larger edits, none have been able to bypass the ratelimiting step of homologous recombination (HR): long-range 5¢ end resection. Here, we describe a novel set of Cas9 fusions, Cas9-HRs, designed to bypass the rate-limiting step of HR r… Show more

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Cited by 15 publications
(10 citation statements)
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“…Therefore, adding DSB end processing enzymes to the cleavage sites is expected to help enhance GT efficiency. Previously reported end resection enzymes and approaches such as hExo1(Hackley, 2021), hHE, CtIP (Charpentier et al, 2018) and T5 exonuclease (T5exo) (Zhang et al, 2020) together with the CRISPR-Cas9 or Cas12a system significantly enhanced homologous recombination-mediated gene editing. We designed and tested GT tools using the end resection enzyme by fusing them to the ttLbCas12a protein (Figure S1) for carrying them to the target sites.…”
Section: Resultsmentioning
confidence: 99%
See 1 more Smart Citation
“…Therefore, adding DSB end processing enzymes to the cleavage sites is expected to help enhance GT efficiency. Previously reported end resection enzymes and approaches such as hExo1(Hackley, 2021), hHE, CtIP (Charpentier et al, 2018) and T5 exonuclease (T5exo) (Zhang et al, 2020) together with the CRISPR-Cas9 or Cas12a system significantly enhanced homologous recombination-mediated gene editing. We designed and tested GT tools using the end resection enzyme by fusing them to the ttLbCas12a protein (Figure S1) for carrying them to the target sites.…”
Section: Resultsmentioning
confidence: 99%
“…The resection of DSBs is necessary for strand annealing and repair by HDR mechanisms. Addition of DSB end processing enzymes has been shown to facilitate HR-mediated repair in mammals (Charpentier et al, 2018; Hackley, 2021; Park et al, 2021; Zhang et al, 2020). However, in our experiments, adding nucleases to targeted sites only changed the profile of the edited products (Figure S2).…”
Section: Discussionmentioning
confidence: 99%
“…Alternatively, increased sealing of the nuclease induced DSB without indel formation, similarly to the results obtained in primary cells (Figure 4F ), might also explain this phenomenon. Interestingly, a recent report showed that fusion of the C-terminal portion of EXO1 to the Cas9 promoted HDR-mediated DSB repair in different cell lines and reduced p53 mediated cytotoxicity ( 37 ). On the contrary, our data show that fusing the full-length EXO1 to Cas9 had no effect on DSB resolution, suggesting that tethering of the entire EXO1 protein to the DSB site impairs precise editing.…”
Section: Discussionmentioning
confidence: 99%
“…Additionally, the cell lines EJ and 5637 were cultured in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 100 U penicillin-streptomycin (Gibco, Gaithersburg, MD, USA) and incubated in a cell culture incubator at 37°C with 5% CO 2 . CTSV-specific knockout was generated in the T24 cells by CRISP/Cas9 mediated gene deletion [ 40 ]. The sgRNA sequence is listed as follows: 5’-GAATACAGCCAAGGGAAACA-3'.…”
Section: Methodsmentioning
confidence: 99%