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

Merker, L.; Schindele, P.; Puchta, H.

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. ... mehr

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Verlagsausgabe §
DOI: 10.5445/IR/1000124099
Veröffentlicht am 16.10.2020
Cover der Publikation
Zugehörige Institution(en) am KIT Botanisches Institut (BOTANIK)
Publikationstyp Zeitschriftenaufsatz
Publikationsmonat/-jahr 09.2020
Sprache Englisch
Identifikator ISSN: 2379-8068
KITopen-ID: 1000124099
Erschienen in Current protocols in plant biology
Band 5
Heft 3
Seiten Art. Nr.: e20117
Vorab online veröffentlicht am 31.08.2020
Schlagwörter CRISPR/Cas, Cas12a, temperature-tolerant
Nachgewiesen in Scopus
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