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

Jia Shou, Jinhuan Li, Qiang Wu
doi: https://doi.org/10.1101/135822
Jia Shou
1Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, SCSB, Shanghai Jiao Tong University (SJTU), Shanghai, 200240, China
2State Key Laboratory of Oncogenes and Related Genes, SJTU Medical School, Shanghai, 200240, China
3School of Life Sciences and Biotechnology, SJTU, Shanghai, 200240, China
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Jinhuan Li
1Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, SCSB, Shanghai Jiao Tong University (SJTU), Shanghai, 200240, China
2State Key Laboratory of Oncogenes and Related Genes, SJTU Medical School, Shanghai, 200240, China
3School of Life Sciences and Biotechnology, SJTU, Shanghai, 200240, China
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Qiang Wu
1Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, SCSB, Shanghai Jiao Tong University (SJTU), Shanghai, 200240, China
2State Key Laboratory of Oncogenes and Related Genes, SJTU Medical School, Shanghai, 200240, China
3School of Life Sciences and Biotechnology, SJTU, Shanghai, 200240, China
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  • For correspondence: qwu123@gmail.com
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Summary

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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 09, 2017.
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Precise and Predictable DNA Fragment Editing Reveals Principles of Cas9-Mediated Nucleotide Insertion
Jia Shou, Jinhuan Li, Qiang Wu
bioRxiv 135822; doi: https://doi.org/10.1101/135822
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Precise and Predictable DNA Fragment Editing Reveals Principles of Cas9-Mediated Nucleotide Insertion
Jia Shou, Jinhuan Li, Qiang Wu
bioRxiv 135822; doi: https://doi.org/10.1101/135822

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