Base Editing with a Cpf1–cytidine Deaminase Fusion
The base editors (BEs) developed by combining APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) or AID (activation-induced deaminase) cytidine deaminase family members with CRISPR-Cas system have been used for targeted C-to-T base editing in various species. These previously developed BEs are dependent on Cas9 proteins for genome targeting, which limit base editing to regions with G/C rich protospacer adjacent motif (PAM) sequences.
Research teams led by Dr. YANG Li at the CAS-MPG Partner Institute for Computational Biology, Chinese Academy of Sciences, Dr. CHEN Jia and Dr. HUANG Xing-Xu at the School of Life Science and Technology, ShanghaiTech University have developed a series of novel CRISPR/Cpf1 (Cas12a) base editors.
In this study, researchers developed a novel BE system based on catalytically dead Lachnospiraceae bacterium Cpf1 (Cas12a).Cpf1 is another Cas protein that differs from Cas9 in several ways: Cpf1 requires a T-rich PAM sequence (TTTV) for target- DNA recognition; the guide RNA for Cpf1 (CRISPR RNA, crRNA) is shorter than that for Cas9 (single guide RNA, sgRNA); and the Cpf1-cleavage site is located distal and downstream relative to the PAM sequence in spacer DNA, rather than proximal and upstream as for Cas9. Cpf1 also induces less off-target cleavage genome wide than does Cas9. These newly developed dCpf1-BEs perform targeted base editing with very low levels of indel formation, non-C-to-T substitutions and off-target editing, thus enabling base editing in A/T-rich regions with high precision.
The paper entitled “Base editing with a Cpf1– cytidine deaminase fusion” was published online in Nature Biotechnologyon March 19, 2018. 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. The deep-sequencing data were generated by the CAS-MPG Partner Institute for Computational Biology Omics Core and have been deposited in the NCBI Gene Expression Omnibus (accession no. GSE110136) and the National Omics Data Encyclopedia (accession no. NODEP00371765) and the National Omics Data Encyclopedia (accession no. NODEP00371765)
Summary of Cas9-based and dCpf1-based BEs (Image by YANG Li's group)