tailieunhanh - Targeted genome modifications in soybean with CRISPR/Cas9

The ability to selectively alter genomic DNA sequences in vivo is a powerful tool for basic and applied research. The CRISPR/Cas9 system precisely mutates DNA sequences in a number of organisms. Here, the CRISPR/ Cas9 system is shown to be effective in soybean by knocking-out a green fluorescent protein (GFP) transgene and modifying nine endogenous loci. | Jacobs et al. BMC Biotechnology 2015 15 16 DOI S12896-015-0131-2 BMC Biotechnology RESEARCH ARTICLE Open Access Targeted genome modifications in soybean with CRISPR Cas9 Thomas B Jacobs1 3 5 Peter R LaFayette2 3 Robert J Schmitz4 and Wayne A Parrott1 2 3 Abstract Background The ability to selectively alter genomic DNA sequences in vivo is a powerful tool for basic and applied research. The CRISPR Cas9 system precisely mutates DNA sequences in a number of organisms. Here the CRISPR Cas9 system is shown to be effective in soybean by knocking-out a green fluorescent protein GFP transgene and modifying nine endogenous loci. Results Targeted DNA mutations were detected in 95 of 88 hairy-root transgenic events analyzed. Bi-allelic mutations were detected in events transformed with eight of the nine targeting vectors. Small deletions were the most common type of mutation produced although SNPs and short insertions were also observed. Homoeologous genes were successfully targeted singly and together demonstrating that CRISPR Cas9 can both selectively and generally target members of gene families. Somatic embryo cultures were also modified to enable the production of plants with heritable mutations with the frequency of DNA modifications increasing with culture time. A novel cloning strategy and vector system based on In-Fusion cloning was developed to simplify the production of CRISPR Cas9 targeting vectors which should be applicable for targeting any gene in any organism. Conclusions The CRISPR Cas9 is a simple efficient and highly specific genome editing tool in soybean. Although some vectors are more efficient than others it is possible to edit duplicated genes relatively easily. The vectors and methods developed here will be useful for the application of CRISPR Cas9 to soybean and other plant species. Keywords CRISPR Cas9 Plant transformation Soybean Genomic engineering Gene targeting Hairy roots Background Methods to specifically target and modify DNA .