CRISPR/CAS9-Mediated Knockout Of BRANCHED1 And BRANCHED2 Gene In Rapeseed Regulates Shoot Branching

Plant architecture depends on shoot branching patterns.The improvement of shoot branching patterns is an efficient way of improving crop yield.However,the molecular mechanism of shoot branching is not properly understood in rapeseed?Brassica napus L.?.Also,genetic resources for the improvement of shoot branching in agricultural crops are lacking.To improve shoot branching traits in plants,the establishment,and utilization of shoot branching gene mutant collections will be required for a better understanding of mechanism regulating shoot branching in B.napus.The CRISPR/Cas9 genome editing technology offers a potent approach to generate a novel allelic variation.In the present study,we demonstrate the successful application of CRISPR/Cas9genome editing technology for the targeted mutations of two shoot branching genes,Bna BRC1and Bna BRC2,in rapeseed.The transformed seedlings were generated using the Agrobacterium-mediated transformation and the transgenic plants were selected using PCR.Hi-TOM sequencing analysis was performed to detect the induced mutations in both Bna BRC1 and Bna BRC2 mutant plants at T₀and T₁generation.The induced mutations were heritably transmitted to successive generations?T₀-T₁?,and a variety of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping.All of these detected homozygous mutations at the target sites within Bna BRC1 are predicted to cause frameshift mutations and results in non-functional proteins in the TCP domains,which encode TCP proteins that regulate many biological processes,such as seed germination,shoot branching and lateral organ development.The development of shoot branching phenotype in B.napus was achieved by the targeted mutations in the Bna BRC1 gene with high efficiency in the S3 target site of conserved TCP domain in both A01 and C01 homologous copies of Bna BRC1 gene.In addition,T-DNA-free plants carrying the desired gene modifications were obtained through genetic segregation.We also investigated the putative genomic off-target loci of the designed sg RNA,and no off-target mutations were detected,indicating that the shoot branching phenotype observed in this study was not induced by the occurrence of off-target mutations.Collectively,our results revealed that CRISPR/Cas9 technology was successfully utilized to knock out Bna BRC1 and Bna BRC2 genes in B.napus.Furthermore,the brc1 shoot branching phenotypes obtained in this study revealed the key role of Bna BRC1 gene in the control of shoot branching and its pleiotropic effect in rapeseed shoot architecture,thus providing the first clue to examine the mechanisms involved in the regulation of shoot architecture in B.napus.