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Plant J. 2019 Nov;100(4):863-872. doi: 10.1111/tpj.14478. Epub 2019 Sep 6.

A CRISPR/LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens.

Pu X1, Liu L1,2, Li P1,2, Huo H3, Dong X1, Xie K4, Yang H1,2, Liu L1.

Author information

1
Key Laboratory for Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, and Yunnan Key Laboratory for Wild Plant Resources, Kunming, 650201, China.
2
University of Chinese Academy of Sciences, Beijing, 100049, China.
3
Department of Environmental Horticulture, Mid-Florida Research and Education Center, University of Florida, Gainesville, FL, 32703, USA.
4
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China.

Abstract

Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non-vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co-delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss-of-function mutants of multiple genes from different gene families.

KEYWORDS:

Physcomitrella patens ; CRISPR/Cas12a; CRISPR/Cas9; genome editing; technical advance

PMID:
31350780
DOI:
10.1111/tpj.14478

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