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SJTU Team Made Disruptive Breakthrough in Gene Editing Mechanisms

July 20, 2018      Author: Shou Jia, Li Jinhuan

On July 19th, Prof. Wu Qiang's team from the Comparative Biomedical Research Center of Shanghai Center for Systems Biomedicine, SJTU, published online its latest research result on world-renowned journal the Molecular Cell. The essay entitling "Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-mediated Nucleotide Insertion" achieved original innovative progress in gene editing mechanisms and subverted existing understanding about gene editing. It revealed the brand-new cutting mechanism of CRISPR/Cas9's latest gene editing system and its role in chromosomal rearrangement and DNA repair, enabling accurate DNA-fragment editing and predictable base insertion.


Summary: Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5′ overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.

The study was completed by Shanghai Jiao Tong University independently, with Prof. Wu Qiang being the correspondence author and SJTU's doctoral student Shou Jia and its postdoctoral student Li Jinhuan being co-first authors. This study was funded by the National Natural Science Foundation of China and the Ministry of Science and Technology of the People's Republic of China.


Translated by Chen Qianqian   Reviewd by Wang Bingyu