干旱区生态环境知识资源中心(Arid): In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

Arid

DOI	10.1111/tpj.14488
	In planta gene targeting can be enhanced by the use of CRISPR/Cas12a
	Wolter, Felix; Puchta, Holger
通讯作者	Puchta, Holger
来源期刊	PLANT JOURNAL
ISSN	0960-7412
EISSN	1365-313X
出版年	2019
卷号	100 期号:5 页码:1083-1094
英文摘要	The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double-strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell-specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20-80-fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non-homologous end-joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns.
英文关键词	CRISPR Cas genome editing genome instability double-strand break repair homologous recombination RTEL-1 Fanconi anaemia blooms syndrome Arabidopsis thaliana technical advance
类型	Article
语种	英语
国家	Germany
开放获取类型	hybrid
收录类别	SCI-E
WOS记录号	WOS:000486816600001
WOS关键词	DOUBLE-STRAND BREAKS ; HOMOLOGY-DIRECTED REPAIR ; DNA-REPAIR ; ACETOHYDROXYACID SYNTHASE ; ARABIDOPSIS ; RECOMBINATION ; MUTAGENESIS ; REPLICATION ; PROTEIN ; CELLS
WOS类目	Plant Sciences
WOS研究方向	Plant Sciences
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/218043
作者单位	Karlsruhe Inst Technol, Bot Inst, POB 6980, D-76049 Karlsruhe, Germany
推荐引用方式 GB/T 7714	Wolter, Felix,Puchta, Holger. In planta gene targeting can be enhanced by the use of CRISPR/Cas12a[J],2019,100(5):1083-1094.
APA	Wolter, Felix,&Puchta, Holger.(2019).In planta gene targeting can be enhanced by the use of CRISPR/Cas12a.PLANT JOURNAL,100(5),1083-1094.
MLA	Wolter, Felix,et al."In planta gene targeting can be enhanced by the use of CRISPR/Cas12a".PLANT JOURNAL 100.5(2019):1083-1094.