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Efficient in planta gene targeting in Arabidopsis using egg cell‐specific expression of the Cas9 nuclease of Staphylococcus aureus
Author(s) -
Wolter Felix,
Klemm Jeannette,
Puchta Holger
Publication year - 2018
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13893
Subject(s) - cas9 , biology , arabidopsis , gene , nuclease , homologous recombination , genetics , crispr , genome editing , streptococcus pyogenes , genome , gene targeting , dna , microbiology and biotechnology , genomic dna , staphylococcus aureus , mutant , bacteria
Summary Gene targeting ( GT ), the programmed change of genomic sequences by homologous recombination ( HR ), is still a major challenge in plants. We previously developed an in planta GT strategy by simultaneously releasing from the genome a ds DNA donor molecule and creating a double‐stranded break ( DSB ) at a specific site within the targeted gene. Using Cas9 form Streptococcus pyogenes (SpCas9) under the control of a ubiquitin gene promoter, we obtained seeds harbouring GT events, although at a low frequency. In the present research we tested different developmentally controlled promotors and different kinds of DNA lesions for their ability to enhance GT of the acetolactate synthase ( ALS ) gene of Arabidopsis . For this purpose, we used Staphylococcus aureus Cas9 (SaCas9) nuclease and the SpCas9 nickase in various combinations. Thus, we analysed the effect of single‐stranded break ( SSB ) activation of a targeted gene and/or the HR donor region. Moreover, we tested whether DSB s with 5′ or 3′ overhangs can improve in planta GT . Interestingly, the use of the SaCas9 nuclease controlled by an egg cell‐specific promoter was the most efficient: depending on the line, in the very best case 6% of all seeds carried GT events. In a third of all lines, the targeting occurred around the 1% range of the tested seeds. Molecular analysis revealed that in about half of the cases perfect HR of both DSB ends occurred. Thus, using the improved technology, it should now be feasible to introduce any directed change into the Arabidopsis genome at will.