RT Journal
A1 He, Jianjie
A1 Zhang, Kai
A1 Yan, Shuxiang
A1 Tang, Mi
A1 Zhou, Weixian
A1 Yin, Yongtai
A1 Chen, Kang
A1 Zhang, Chunyu
A1 Li, Maoteng
T1 Genome-scale targeted mutagenesis in Brassica napus using a pooled CRISPR library
JF Genome Research 
JO Genome Research 
YR 2023 
FD May 01 
VO 33 
IS 5 
SP 798 
OP 809 
DO 10.1101/gr.277650.123 
UL http://genome.cshlp.org/content/33/5/798.abstract 
AB The recently constructed mutant libraries of diploid crops by the CRISPR-Cas9 system have provided abundant resources for functional genomics and crop breeding. However, because of the genome complexity, it is a big challenge to accomplish large-scale targeted mutagenesis in polyploid plants. Here, we demonstrate the feasibility of using a pooled CRISPR library to achieve genome-scale targeted editing in an allotetraploid crop of Brassica napus. A total of 18,414 sgRNAs were designed to target 10,480 genes of interest, and afterward, 1104 regenerated transgenic plants harboring 1088 sgRNAs were obtained. Editing interrogation results revealed that 93 of the 178 genes were identified as mutated, thus representing an editing efficiency of 52.2%. Furthermore, we have discovered that Cas9-mediated DNA cleavages tend to occur at all the target sites guided by the same individual sgRNA, a novel finding in polyploid plants. Finally, we show the strong capability of reverse genetic screening for various traits with the postgenotyped plants. Several genes, which might dominate the fatty acid profile and seed oil content and have yet to be reported, were unveiled from the forward genetic studies. Our research provides valuable resources for functional genomics, elite crop breeding, and a good reference for high-throughput targeted mutagenesis in other polyploid plants.