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APOBEC3 Induces Mutations During Repair of CRISPR/Cas9-generated DNA Breaks

Jan 02, 2018     Email"> PrintText Size

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system, which provides so far the most convenient and efficient genome editing, has been broadly applied in life science research and shows great potential in clinics to treat human diseases. Due to the risk of permanent and inherited off-target/unintended editing in genomic DNA, scientists have strived to improve CRISPR technology, aiming to increase its fidelity and accuracy.

Dr. YANG Li's group at the CAS-MPG Partner Institute for Computational Biology of Chinese Academy of Sciences, collaborating with researchers at ShanghaiTech University and Nanjing Medical University demonstrated the underlying mechanism of mutagenesis during repair of CRISPR/Cas9-generated DNA breaks, providing new strategies to increase the fidelity and accuracy of CRISPR/Cas9-mediated genome editing. The study was published in Nature Structural & Molecular Biology.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like/activation-induced cytidine deaminase (APOBEC/AID) family of proteins, prefers single-stranded nucleic acids for cytidine to uracil deamination, which are commonly involved in DNA repair system for the breaks generated by CRISPR/Cas9. It is intriguing to examine whether APOBECs can target these single-stranded nucleic acids to affect the repair outcome of CRISPR/Cas9-generated DNA breaks. 

In this study, the researchers showed that APOBEC3 can trigger cytidine deamination of single-stranded oligodeoxynucleotides (ssODNs), one kind of single-stranded nucleic acids, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair of Cas9-generated double-strand breaks.

In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels). Both APOBEC3-mediated deamination and DNA-repair proteins play important roles in the generation of these indels.

Based on these findings, researchers suggested to use double-stranded ODNs (dsODNs), instead of ssODN, as homologous donors for high-fidelity gene correction in postnatal tissues or organs, and to temporarily suppress endogenous APOBEC3s to repress these unwanted mutations in genomic DNA.

This study was supported by the grants from National Natural Science Foundation of China, Ministry of Science and Technology, Shanghai Municipal Science and Technology Commission and ShanghaiTech University.

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(Editor: LIU Jia)

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