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Title: Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality

Alfalfa (Medicago sativaL.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting theCOUMARATE 3-HYDROXYLASE (MsC3H)gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon ofMsC3Hwere designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa viaAgrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles ofMsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, andMsc3h-158) with different mutation events inMsC3Hwere characterized in detail. Homozygous mutation ofMsC3Hin these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, andin vitrotrue dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing ofMsC3Hsuccessfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

 
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Award ID(s):
2233714
NSF-PAR ID:
10507214
Author(s) / Creator(s):
; ; ; ; ; ; ; ;
Publisher / Repository:
Frontier Media SA
Date Published:
Journal Name:
Frontiers in Plant Science
Volume:
15
ISSN:
1664-462X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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