Recently, the international authoritative journal Plant Biotechnology Journal published a research paper entitled "CRISPR-BETS: A base editing design tool for generating stop codons" online, which elaborates the use of single-base editing technology to accurately knock out target genes, and develops a research paper called CRISPR-BETS (base editing to stop) gRNA design software.
Using Cytosine base editors (CBEs) single-base editing technology, the base of the target site can be accurately replaced, and the stop codon can be generated, thereby achieving knockout of the target gene. Compared with the traditional Cas9 knockout technology, the use of CBEs gene knockout can not change the genome size, accurately obtain the expected editing results, most of the genes in the plant can be knocked out by CBEs technology, and the homozygous mutants can be obtained in the first generation of edited plants, which greatly reduces the workload of late screening, and the technology has great application value.
Designing accurate and rigorous gRNAs is the first step in using CBEs systems for efficient and precise knockout of genes. To this end, the authors developed CRISPR-BETS (https://zhangtaolab.org/software/crisprbets) gRNA design software focusing on plants, which can design appropriate gRNAs according to different cytosine deaminase characteristics, Cas9 variants, PAM and editing windows, and improve editing efficiency by reducing the off-target ratio through gRNA-specific screening and optimization. CRISPR-BETS software is simple to operate and user-friendly, and can be used directly without the need for experimenters to master complex bioinformatics knowledge, and the software only needs to enter GenBank, Snapgene or Fasta format sequence files to complete gRNA design. At present, the software has supported gRNA design and specific optimization analysis of 91 plant species and other model species such as humans, mice, zebrafish and so on.
Figure 1: CRISPR-BETS workflow
In order to verify the reliability of the CRISPR-BETS software, the authors first used the rice and tomato protoplast editing systems to knock out the genes of OsGW2, OsPDS, SlBlc, etc., respectively, and verified by amplicon sequencing that CBEs can efficiently generate early termination codons, thereby knocking out the target gene. At the same time, the authors evaluated the efficiency of the strategy in stable transformation, and the results showed that the editing efficiency of the system was high, and the homozygous mutant strains could be reliably generated in the first generation of editing.
Figure 2: Protoplasts and stable transformation evaluation CBEs knockout systems
Professor Zhang Tao of Yangzhou University, Professor Daniel Zhang of university of electronic science and technology of China and professor Qi Yiping of the University of Maryland as co-corresponding authors of this paper, Wu Yuechao, doctoral candidate of Yangzhou University, and He Yao, doctoral candidate of University of Electronic Science and Technology of China, are the co-first authors of this paper, and the research work has been funded by the National Natural Science Foundation of China, the Innovative Talents of Sichuan Provincial Science and Technology Department, the Open Project of Jiangsu Key Laboratory of Crop Genetics and Physiology, and the Plant Genome Research Program of the National Science Foundation of the United States.
Original link:
https://onlinelibrary.wiley.com/doi/abs/10.1111/pbi.13732
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