Author:
Mehryar Mohammadreza M.,Shi Xin,Li Jingwei,Wu Qiang
Abstract
AbstractRecent studies have shown that Cas9-mediated CRISPR gene editing outcomes at junctions of chromosomal rearrangements are precise and predictable; however, the underlying mechanisms are poorly understood due to lack of suitable assay system and analysis tool. Here we developed a customized computer program to take account of staggered Cas9 cleavage and to rapidly process large volumes of junctional sequencing reads from chromosomal rearrangements, including DNA-fragment inversions, duplications, and deletions. We also established a sensitive assay system usingHPRT1andDCKas reporters for cell growth during DNA-fragment editing by Cas9 with dual sgRNAs and found prominent large resections at junctions of chromosomal rearrangements. In addition, we found that knockdown ofPolQ(encoding Polθ) results in increased large resections but decreased small deletions. We also found that the mechanisms for generating small deletions of 1bp and >1bp during DNA-fragment editing are different with regards to their opposite dependencies on Polθ and Polλ. Specifically, Polθ suppresses 1bp deletions but promotes >1bp deletions, whereas Polλ promotes 1bp deletions but suppresses >1bp deletions. Finally, we found that Polλ is the main DNA polymerase responsible for fill-in of the 5’ overhangs of staggered Cas9 cleavage ends. These findings contribute to our understanding of the molecular mechanisms of CRISPR/Cas9-mediated DNA-fragment editing and have important implications for controllable, precise, and predictable gene editing.
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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