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Title: High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR /Cas9
Summary

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved usingAgrobacteriumT‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such asNicotinana tabacum(tobacco) andSolanum lycopersicum(tomato), greater than 10‐fold enhancements inGTfrequencies have been achieved usingDNAvirus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundantSSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carryingCRISPR/Cas9 nucleases and repair templates achievedGTat an endogenousubiquitinlocus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these highGTfrequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with theWDVreplicons, multiplexedGTwithin the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies ofGTusingWDV‐basedDNAreplicons will make it possible to edit complex cereal genomes without the need to integrateGTreagents into the genome.

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NSF-PAR ID:
10033348
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
The Plant Journal
Volume:
89
Issue:
6
ISSN:
0960-7412
Page Range / eLocation ID:
p. 1251-1262
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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