TY - JOUR T1 - Precise and Predictable DNA Fragment Editing Reveals Principles of Cas9-Mediated Nucleotide Insertion JF - bioRxiv DO - 10.1101/135822 SP - 135822 AU - Jia Shou AU - Jinhuan Li AU - Qiang Wu Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/05/09/135822.abstract N2 - 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. ER -