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. 2021 Aug;64(8):1355-1367.
doi: 10.1007/s11427-020-1775-2. Epub 2021 Jan 6.

Efficient and high-fidelity base editor with expanded PAM compatibility for cytidine dinucleotide

Zhiquan Liu #  1 , Siyu Chen #  1 , Yingqi Jia  1 , Huanhuan Shan  1 , Mao Chen  1 , Yuning Song  1 , Liangxue Lai  2   3   4   5 , Zhanjun Li  6
Affiliations

Efficient and high-fidelity base editor with expanded PAM compatibility for cytidine dinucleotide

Zhiquan Liu et al. Sci China Life Sci. 2021 Aug.

Abstract

Cytidine base editor (CBE), which is composed of a cytidine deaminase fused to Cas9 nickase, has been widely used to induce C-to-T conversions in a wide range of organisms. However, the targeting scope of current CBEs is largely restricted to protospacer adjacent motif (PAM) sequences containing G, T, or A bases. In this study, we developed a new base editor termed "nNme2-CBE" with excellent PAM compatibility for cytidine dinucleotide, significantly expanding the genome-targeting scope of CBEs. Using nNme2-CBE, targeted editing efficiencies of 29.0%-55.0% and 17.3%-52.5% were generated in human cells and rabbit embryos, respectively. In contrast to conventional nSp-CBE, the nNme2-CBE is a natural high-fidelity base editing platform with minimal DNA off-targeting detected in vivo. Significantly increased efficiency in GC context and precision were determined by combining nNme2Cas9 with rationally engineered cytidine deaminases. In addition, the Founder rabbits with accurate single-base substitutions at Fgf5 gene loci were successfully generated by using the nNme2-CBE system. These novel nNme2-CBEs with expanded PAM compatibility and high fidelity will expand the base editing toolset for efficient gene modification and therapeutic applications.

Keywords: CRISPR; N4CC PAM; Nme2Cas9; base editor.

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References

    1. Agudelo, D., Carter, S., Velimirovic, M., Duringer, A., Rivest, J.F., Levesque, S., Loehr, J., Mouchiroud, M., Cyr, D., Waters, P.J., et al. (2020). Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9. Genome Res 30, 107–117. - PubMed - PMC - DOI
    1. Amrani, N., Gao, X.D., Liu, P., Edraki, A., Mir, A., Ibraheim, R., Gupta, A., Sasaki, K.E., Wu, T., Donohoue, P.D., et al. (2018). NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome Biol 19, 214. - PubMed - PMC - DOI
    1. Anzalone, A.V., Randolph, P.B., Davis, J.R., Sousa, A.A., Koblan, L.W., Levy, J.M., Chen, P.J., Wilson, C., Newby, G.A., Raguram, A., et al. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157. - PubMed - PMC - DOI
    1. Bae, S., Park, J., and Kim, J.S. (2014). Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475. - PubMed - PMC - DOI
    1. Billon, P., Bryant, E.E., Joseph, S.A., Nambiar, T.S., Hayward, S.B., Rothstein, R., and Ciccia, A. (2017). CRISPR-mediated base editing enables efficient disruption of eukaryotic genes through induction of STOP codons. Mol Cell 67, 1068–1079.e4. - PubMed - PMC - DOI

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