Increasing Cytosine Base Editing Scope and Efficiency With Engineered Cas9-PmCDA1 Fusions and the Modified sgRNA in Rice

Conclusion In this study, we described two efficient PmCDA1-based CBE systems, SpCas9n-pBE and VQRn-pBE, that will help to expand the scope of cytosine base editing in rice. The effective deamination window typically spanned positions 1–7 of the protospacer and the target single C showed the highest editing frequency at or near position 3. The mutant genotypes were mainly single or double C-to-T substitutions. Furthermore, the editing efficiency of VQRn-pBE was increased by the modified sgRNA. These base editors will be useful tools for scientific research and crop breeding in rice. Author Contributions JY, CZ, and YW designed the experiments and wrote the manuscript. YW, FW, SZ, FF, and JS performed all the experiments. WX and FW analyzed the results. JY supervised the project. All authors read and approved the final manuscript. Conflict of Interest Statement The authors submitted a patent application based on the results reported in this paper. Supplementary Material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2019.00379/full#supplementary-material Abbreviations Cas9, CRISPR-associated protein 9; CBE, cytosine base editor; CRISPR, clustered regularly interspaced short palindromic repeats; C-to-T, cytosine-to-thymine; EQR, engineered SpCas9 variant with residue substitutions D1135E/R1335Q/T1337R; Indel, insertion and deletion; PAM, protospacer adjacent motif; pBE, PmCDA1-based cytosine...
Source: Frontiers in Genetics - Category: Genetics & Stem Cells Source Type: research