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Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana CRISPR/Cas systems enable gene editing through the induction of site‐specific DNA double‐strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non‐homologous end joining (NHEJ)‐mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T‐rich regions. Most recently, we tested an improved, temperature‐tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Protocols in Plant Biology Wiley

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

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Publisher
Wiley
Copyright
© 2020 Wiley Periodicals LLC
ISSN
2379-8068
eISSN
2379-8068
DOI
10.1002/cppb.20117
Publisher site
See Article on Publisher Site

Abstract

CRISPR/Cas systems enable gene editing through the induction of site‐specific DNA double‐strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non‐homologous end joining (NHEJ)‐mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T‐rich regions. Most recently, we tested an improved, temperature‐tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors.

Journal

Current Protocols in Plant BiologyWiley

Published: Sep 1, 2020

Keywords: ; ;

References