Chen W.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources |
Chen W.,Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions |
Chen W.,South China Agricultural University |
Zeng D.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources |
And 19 more authors.
Biotechnology and Biotechnological Equipment | Year: 2016
Cloning of coding sequence (CDS) is an important step for gene function research. Here, we reported a simple and efficient strategy for assembling multiple-exon into an intron-free CDS from genomic DNA (gDNA) by an isothermal recombination reaction-based PCR (IRR-PCR) method. As an example, a 2067-bp full-length CDS of the anther-specific expression gene OsABCG15, which is composed of seven exons and six introns, was generated by IRR-PCR using genomic DNA of rice leaf as the template. Actually, this approach can be wildly applied to any DNA sequences assembly to achieve CDS cloning, gene fusion and multiple site-directed mutagenesis in functional genomics studies in vitro. © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
Ma X.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources |
Ma X.,South China Agricultural University |
Zhang Q.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources |
Zhang Q.,Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions |
And 49 more authors.
Molecular Plant | Year: 2015
Abstract CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement. © 2015 The Author.