DNA Polymerases in Precise and Predictable CRISPR/Cas9-mediated Chromosomal Rearrangements

Recent 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 using HPRT1 and DCK as 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 of PolQ (encoding Pol{theta}) 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 Pol{theta} and Pol{lambda}. Specifically, Pol{theta} suppresses 1bp deletions but promotes >1bp deletions, whereas Pol{lambda} promotes 1bp deletions but suppresses >1bp deletions. Finally, we found that Pol{lambda} 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.