Abstract:
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[EN] Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas ...[+]
[EN] Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.
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Project ID:
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info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2017-83184-R/ES/VIRUS DE PLANTAS: PATOGENOS Y TAMBIEN VECTORES PARA LA PRODUCCION DE PROTEINAS, METABOLITOS, RNAS Y NANOPARTICULAS/
info:eu-repo/grantAgreement/MICINN//BES-2017-0890098/
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-108203RB-I00/ES/EXPLOITING THE MODULAR ARCHITECTURE OF CRISPR%2FCAS TO DESIGN PROGRAMMABLE GENE CIRCUITS AND OTHER NEW BREEDING TOOLS IN PLANTS/
info:eu-repo/grantAgreement/MICINN//FPU17%2F05503/
info:eu-repo/grantAgreement/EC/H2020/760331/EU
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Thanks:
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This work was supported by grants BIO2017-83184-R and PID2019-108203RB-I00 from Ministerio de Ciencia e Innovacion (Spain) through the Agencia Estatal de Investigacion (co-financed European Regional Development Fund), and ...[+]
This work was supported by grants BIO2017-83184-R and PID2019-108203RB-I00 from Ministerio de Ciencia e Innovacion (Spain) through the Agencia Estatal de Investigacion (co-financed European Regional Development Fund), and H2020-760331 Newcotiana from the European Commission. M.U. and S.S. are the recipients of fellowships FPU17/05503 and BES-2017-0890098, respectively, from Ministerio de Ciencia e Innovacion (Spain)
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