Expanding target editing space in the wheat genome using the variants of the Cas12a and Cas9 nucleases

Authors: WANG, WEI - Kansas State University; TIAN, BIN - Kansas State University; PAN, QIANLI - Kansas State University; CHEN, YUEYING - Kansas State University; HE, FEI - Kansas State University; Bai, Guihua; AKHUNOVA, ALINA - Kansas State University; TRICK, HAROLD - Kansas State University; AKHUNOV, EDUARD - Kansas State University

Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2021
Publication Date: 12/9/2020
Citation: Wang, W., Tian, B., Pan, Q., Chen, Y., He, F., Bai, G., Akhunova, A., Trick, H., Akhunov, E. 2020. Expanding target editing space in the wheat genome using the variants of the Cas12a and Cas9 nucleases. Plant Biotechnology Journal. https://doi.org/10.1111/pbi.13669.
DOI: https://doi.org/10.1111/pbi.13669

Interpretive Summary: The development of new CRISPR-based gene editing technologies could broaden the range of their applications in wheat. Here we evaluated the editing efficiency of different technologies including two versions of Cas12a (FnCas12a and LbCas12a) and one of Cas9, each having different recognition specificities. LbCas12a induced mutations in the wheat genome although at a lower rate than Cas9, but FnCas12a did not. The editing efficiency of LbCas12a increased eight-fold by using guide RNAs flanked by ribozymes and driven by the RNA polymerase II promoter. LbCas12a showed similar efficiency between multiplexed genome editing (MGE) and the simplex editing. Multiplexed genome editing using LbCas12a successfully changed grain size and weight in wheat. Therefore, the set of editable target loci in the wheat genome could be expanded by using these new CRISPR-based tools.

Technical Abstract: The development of CRISPR-based editors having different protospacer adjacent motif (PAM) recognition specificities, or guide RNA length/structure requirements broadens the range of possible genome editing applications. Here, we evaluated the natural and engineered variants of Cas12a (FnCas12a from Francisella novicida and LbCas12a from Lachnospiraceae bacterium) and Cas9 for wheat genome editing efficiency and ability to create heritable mutations in genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a was able to induce mutations in the wheat genome in the current study, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency of LbCas12a was achieved by using the guide RNAs flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides. The LbCas12a-MGE construct was applied for generating heritable mutations in genes controlling grain size and weight. The range of editable target loci in the wheat genome could be expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9.