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| Criteria to design efficient sgRNAs instituted |
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With the rapid development of genome sequencing technology and bioinformatics, increasing genome-scale genetic elements are annotated in different species. The CRISPR (clustered regularly interspaced short palindromic repeat)-associated (Cas) endonuclease Cas9 (CRISPR/Cas9-sgRNA) mediated precise genome editing is being universally applied in diverse plant species. A major concern of CRISPR/Cas9-sgRNA system is the editing efficiency and specificity of single guide RNA (sgRNA). Genome editing in plants takes long time to obtain transgenic products. Thus, criteria that can be used to distinguish efficient and inefficient sgRNAs are of great utility for avoiding generation of non-edited transgenic plants resulting from inefficient sgRNAs.
Prof. YU Diqiu’s team of Xishuangbanna Tropical Botanical Garden (XTBG) instituted criteria for efficient sgRNAs based on nucleotide composition of guide sequences and secondary structure of sgRNAs. They also introduced a new strategy to construct multiple target editing CRISPR/Cas9-sgRNA cassettes.
The researchers instituted criteria for selection of efficient sgRNAs. Firstly, CRISPR/Cas9-sgRNA content between 30% and 80%; secondly, intact secondary structures except for stem loop 1; thirdly, no more than 12 total base pairs (TBPs) and no more than 7 consecutive base pairs (CBPs); and finally, no more than 6 internal base pairs (IBPs).
They then developed a restriction enzyme based system which consisted of two modules for the assembly of multiple sgRNA cassettes and the Cas9 gene to assemble Cas9 and multiple sgRNAs into a T-DNA.
Having instituted the criteria for selection of efficient sgRNAs in plants, subsequently the researchers performed experimental test in rice plants. The results demonstrated that the clone strategy for assembly of multiple sgRNAs was rapid and functional, and the criteria could be used to select efficient sgRNAs from the highly specific sgRNAs.
Their toolbox for sgRNA design criteria and assembly of multiplex CRISPR/Cas9-sgRNA system provided researchers with a new approach to efficiently edit one or multiple target sites and perform genetic improvement.
The study entitled “Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing” has been published in Scientific Reports.
Contact
YU Diqiu, Ph.D Principal Investigator
Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Kunming, Yunnan 650223, China Tel: 86 871 65178133
E-mail: ydq@xtbg.ac.cn |
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