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Precise and Predictable DNA Fragment Editing Reveals Principles of Cas9-Mediated Nucleotide Insertion

56 Pages Posted: 13 Apr 2018 Sneak Peek Status: Review Complete

See all articles by Jia Shou

Jia Shou

Shanghai Jiao Tong University (SJTU) - Center for Comparative Biomedicine

Jinhuan Li

Shanghai Jiao Tong University (SJTU) - Center for Comparative Biomedicine

Qiang Wu

Shanghai Jiao Tong University (SJTU) - Center for Comparative Biomedicine

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Abstract

DNA fragment editing (DFE) or chromosomal engineering including inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate structural genome variations and developmental gene regulation, but little is known about the underlying mechanisms. Here we report that debilitating CtIP, which is thought to function in NHEJ, enhances precise DNA fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA fragment inversions, deletions, and duplications, we find that Cas9 cleaves the noncomplementary strand with a flexible profile upstream of the PAM site and rationally-designed Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions of DFE are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DFEs could be achieved by perturbing DNA repair genes and using appropriate PAM configurations. These findings have important applications regarding 3D chromatin folding and enhancer insulation during gene regulation.

Suggested Citation

Shou, Jia and Li, Jinhuan and Wu, Qiang, Precise and Predictable DNA Fragment Editing Reveals Principles of Cas9-Mediated Nucleotide Insertion (2018). Available at SSRN: https://ssrn.com/abstract=3155807 or http://dx.doi.org/10.2139/ssrn.3155807
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Jia Shou

Shanghai Jiao Tong University (SJTU) - Center for Comparative Biomedicine

Shanghai, 200240
China

Jinhuan Li

Shanghai Jiao Tong University (SJTU) - Center for Comparative Biomedicine

Shanghai, 200240
China

Qiang Wu (Contact Author)

Shanghai Jiao Tong University (SJTU) - Center for Comparative Biomedicine ( email )

Shanghai, 200240
China

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