A simple and reliable method for creating PCR-detectable mutants in Arabidopsis with the polycistronic tRNA-gRNA CRISPR/Cas9 system

• Autorzy: Hui L. , Zhao M. , He J. , Hu Y. , Huo Y. , Hao H. , Hao Y. , Zhu W. , Wang Y. , Xu M. , Fu A.
• Języki publikacji: EN

Abstrakty

EN

To develop an easy and robust method for creating genetically stable and easily detectable Arabidopsis mutants, we adopted the polycistronic tRNA–gRNA CRISPR/Cas9 (PTG/Cas9) system, a multiplex gene-editing tool in rice, with PTOX as the reporter gene. The PTG/Cas9 system has a great potential in generating large deletions detectable by PCR, which greatly simplifies the laborious work of mutant screening. We constructed a PTOX–PTG/Cas9 system with five gRNAs and introduced it into Arabidopsis. At T1 generation, 24.4% of transgenic plants were chimeric with PCR-detectable deletions in PTOX locus, but no homozygous mutant was found, indicating that gene editing occurred predominantly in somatic cells. After a self-cross propagation, 60% of T1 chimeric plants were able to produce homozygous, heterozygous, or bi-allelic ptox offsprings. Inheritable homozygous ptox mutants without Cas9 gene can be obtained earliest at T2 generation. We further targeted five other genes using the same procedure and achieved homozygous Cas9-free mutants with large deletions for all genes within three generations. We established a standard and reliable protocol to generate stable inherited deletion mutants in 2–3 generations along with simple PCR screening methods. We conclude that the rice PTG/Cas9 system is an efficient, easy, and rapid tool to edit genes in Arabidopsis. We propose that it could be applied to other genes in Arabidopsis, and it might have the potential to edit genes in other plant species as well.

• Wydawca: -
• Czasopismo: Acta Physiologiae Plantarum
• Rocznik: 2019
• Tom: 41
• Numer: 10
• Opis fizyczny: Article 170 [14p.], fig.,ref.

Twórcy

autor

Hui L.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

Zhao M.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

He J.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

Hu Y.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

Huo Y.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

Hao H.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

Hao Y.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

Zhu W.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

Wang Y.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

Xu M.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

autor

Fu A.

• Chinese Education Ministry’s Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University (Xi’an), 229 North Taibai Road, Xi’an, Shaanxi 710069, China

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Identyfikatory

DOI

10.1007/s11738-019-2961-3