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BMC Biotechnology

, 15:16

First Online: 12 March 2015Received: 26 September 2014Accepted: 27 February 2015DOI: 10.1186-s12896-015-0131-2

Cite this article as: Jacobs, T.B., LaFayette, P.R., Schmitz, R.J. et al. BMC Biotechnol 2015 15: 16. doi:10.1186-s12896-015-0131-2

Abstract

BackgroundThe 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.

ResultsTargeted 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.

ConclusionsThe 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.

KeywordsCRISPR-Cas9 Plant transformation Soybean Genomic engineering Gene targeting Hairy roots AbbreviationsGFPGreen-fluorescent protein

CRISPRClustered, regularly interspaced, short palindromic repeats

CasCRISPR associated

gRNAGuide RNA

ntnucleotide

DSBDouble-strand break

NHEJNon-homologous end joining

SNPSingle nucleotide polymorphism

miRNAsmicroRNAs

bpBase pair

ZFNsZinc-finger nuclease

TALENsTranscription activator-like effector nucleases

nosnopaline synthase

hphhygromycin phosphotransferase

PAMProtospacer-adjacent motif

Electronic supplementary materialThe online version of this article doi:10.1186-s12896-015-0131-2 contains supplementary material, which is available to authorized users.

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Autor: Thomas B Jacobs - Peter R LaFayette - Robert J Schmitz - Wayne A Parrott

Fuente: https://link.springer.com/







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