CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean

Abstract

At present, the application of CRISPR/Cas9 in soybean (<i>Glycine max</i> (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in <i>GmFT2a</i> (Glyma16g26660) and <i>GmFT5a</i> (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in <i>GmFT2a</i>. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in <i>GmFT2a</i> and 15.8% for target fragments ranging from 1069 to 1161 bp in <i>GmFT5a</i>. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 'transgene-free' homozygous <i>ft2a</i> mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean.