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2000
Volume 1, Issue 1
  • ISSN: 2210-2981
  • E-ISSN: 2210-2914

Abstract

Background: CRISPR-Cas9 is a powerful technology that allows us to modify DNA sequences in a specific manner across a variety of organisms. Due to its high efficiency and specificity, and ease of use, it becomes a commonly used method for gene editing. Although many structural and biochemical studies have been carried out to understand the fundamental mechanism of CRISPR/Cas9, our understanding of CRISPR/Cas9 caused off-target effects is still lacking. Methods: The enhanced in vitro cleavage activity of Cas9 protein from Streptococcus pyogenes (SpCas9) was evaluated by both synthetic crRNA-tracrRNA duplexes and in vitro transcribed single guide RNAs. Results: Here, we report an unexpected finding that mismatches between the guide RNA and target DNA significantly enhanced the in vitro cleavage activity of SpCas9 by more than 2 folds. Conclusion: Our observation that mismatches between the guide RNA and target DNA can dramatically increase the in vitro cleavage of Cas9 suggests the potential sequence preference for the CRSIPR/Cas9 system.

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/content/journals/ccs/10.2174/2210298101999200907161320
2021-01-01
2025-06-07
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  • Article Type:
    Other
Keyword(s): Cas9; CRISPR; endonuclease; guide RNA; mismatch; off target
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