Title:In vitro Assay Revealed Mismatches between Guide RNA and Target DNA can Enhance Cas9 Nuclease Activity
Volume: 1
Issue: 1
Author(s): Ji Luan, Zhen Li, Hailong Wang*, Jun Fu and Youming Zhang
Affiliation:
- State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Helmholtz International Lab for Anti-infectives, Shandong University-Helmholtz Institute of Biotechnology, Shandong University, Qingdao, Shandong, 266237,China
Keywords:
CRISPR, Cas9, guide RNA, mismatch, off target, endonuclease.
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.
