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Highly Efficient base editing in Viral Genome based on Bacterial Artificial Chromosome Using a Cas9-Cytidine Deaminase Fused Protein

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Virologica Sinica 2020年第2期35卷 191-199页

作者： Ke Zheng Fang-Fang Jiang Le Su Xin Wang Yu-Xin Chen Huan-Chun Chen Zheng-Fei Liu State Key Laboratory of Agricultural Microbiology and Key Laboratory of Preventive Veterinary Medicine in Hubei Province.College of Veterinary Medicine.Huazhong Agricultural University Wuhan 430070China Gene editing Research Center Hebei University of Science and Technology.Shijiazhuang 050018China

Viruses evolve rapidly and continuously threaten animal health and economy,posing a great demand for rapid and efficient genome editing technologies to study virulence mechanism and develop effective vaccine.We present a highly efficient viral genome manipulation method using CRISPR-guided cytidine deaminase.We cloned pseudorabies virus genome into bacterial artificial chromosome,and used CRISPR-guided cytidine deaminase to directly convert cytidine(C)to uridine(U)to induce premature stop mutagenesis in viral genes.The editing efficiencies were 100%.Comprehensive bioinformatic analysis revealed that a large number of editable sites exist in pseudorabies virus(PRV)genomes.Notably,in our study viral genome exists as a plasmid in E.coli,suggesting that this method is virus species-independent.This application of base-editing provided an alternative approach to generate mutant virus and might accelerate study on virulence and vaccine development.

关键词： Pseudorabies virus(PRV) Bacterial artificial chromosome(BAC) base-editing CRISPR7Cas9 Genome editing

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Efficient generation of targeted point mutations in the Brassica oleracea var.botrytis genome via a modified CRISPR/Cas9 system

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Horticultural Plant Journal 2022年第4期8卷 527-530页

作者： Guixiang Wang Mei Zong Di Liu Yage Wu Shouwei Tian Shuo Han Ning Guo Mengmeng Duan Liming Miao Fan Liu Beijing Vegetable Research Center Beijing Academy of Agriculture and Forestry SciencesKey Laboratory of Biology and Genetic Improvement of Horticultural Crops(North China)Ministry of AgricultureBeijing Key Laboratory of Vegetable Germplasm ImprovementBeijing 100097China

In this study,we used the modified CRISPR/Cas9 system to produce targeted point mutations in cauliflower.Acetolactate synthase(ALS)and Centromere-specific histone H3 variant(CENH3)genes were selected as the base-editing targets and hypocotyls of cauliflower were used as explants.For ALS gene,a C-to-T conversion in the Pro182 codon(CCT)can alter the encoded amino acid,likely resulting in herbicide resistance,and a C-to-T mutation in the Leu133 codon(CTT)in the CENH3 gene may produce a haploid inducer.Results indicated that the transformation efficiency was 1.8%–4.5%and the mutation efficiencies for the ALS and CENH3 genes were approximately 22%and 87%,respectively.The ALS mutant cauliflower showed strong herbicide resistance,with possible immediate implications for broadleaf weed control in cauliflower fields.

关键词： Cauliflower Targeted point mutations base-editing CRISPR/Cas9 ALS CENH3

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