Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis.

Mao, Yanfei; Zhang, Zhengjing; Feng, Zhengyan; Wei, Pengliang; Zhang, Hui; Botella, José Ramón; Zhu, Jian-Kang

Mao, Yanfei; Zhang, Zhengjing; Feng, Zhengyan; Wei, Pengliang; Zhang, Hui; Botella, José Ramón; Zhu, Jian-Kang.

Afiliación

Mao Y; Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.
Zhang Z; Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.
Feng Z; University of Chinese Academy of Sciences (CAS), Shanghai, China.
Wei P; Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.
Zhang H; University of Chinese Academy of Sciences (CAS), Shanghai, China.
Botella JR; Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Zhu JK; Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, China.

Plant Biotechnol J ; 14(2): 519-32, 2016 Feb.

Article en En | MEDLINE | ID: mdl-26360626

RESUMEN

The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.

Asunto(s)

Arabidopsis/genética; Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética; Ingeniería Genética/métodos; Células Germinativas/metabolismo; Patrón de Herencia/genética; Secuencia de Bases; Quimera; Cruzamientos Genéticos; Genes de Plantas; Vectores Genéticos/metabolismo; Técnicas de Genotipaje; Células Germinativas de las Plantas/metabolismo; Hibridación Genética; Mutagénesis Sitio-Dirigida; Mutación/genética; Especificidad de Órganos/genética; Regiones Promotoras Genéticas/genética

Palabras clave

Arabidopsis; CRISPR-Cas9; gene modification; germ-line-specific

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ingeniería Genética / Arabidopsis / Patrón de Herencia / Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas / Células Germinativas Idioma: En Revista: Plant Biotechnol J Asunto de la revista: BIOTECNOLOGIA / BOTANICA Año: 2016 Tipo del documento: Article País de afiliación: China Pais de publicación: Reino Unido

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