Efficient protein tagging and cis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants.

Autor:	Kumar J; Department of Plant and Microbial Biology, University of Minnesota, USA.; Center for Precision Plant Genomics, University of Minnesota, USA., Char SN; Division of Plant Sciences and Technology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA., Weiss T; Department of Plant and Microbial Biology, University of Minnesota, USA.; Center for Precision Plant Genomics, University of Minnesota, USA., Liu H; Division of Plant Sciences and Technology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA., Liu B; Division of Plant Sciences and Technology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA., Yang B; Division of Plant Sciences and Technology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.; Donald Danforth Plant Science Center, St. Louis, MO 63132, USA., Zhang F; Department of Plant and Microbial Biology, University of Minnesota, USA.; Center for Precision Plant Genomics, University of Minnesota, USA.
Jazyk:	angličtina
Zdroj:	The Plant cell [Plant Cell] 2023 Aug 02; Vol. 35 (8), pp. 2722-2735.
DOI:	10.1093/plcell/koad139
Abstrakt:	Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient nonhomologous end-joining-mediated targeted insertion method was recently developed by combining clustered regularly interspaced short palindromic repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated nuclease 9 (SpCas9) gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet, this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5' overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5' overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis) and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted 2 distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner. Competing Interests: Conflict of interest statement. None declared. (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)
Databáze:	MEDLINE
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