Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China.
The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China.
Int J Mol Sci. 2023 Aug 28;24(17):13331. doi: 10.3390/ijms241713331.
The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin () variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the gene. We found that mutating four consecutive G bases in the intron-2 of the gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the gene.
日本脑炎病毒（JEV）是一种蚊媒黄病毒，可导致亚洲和西太平洋地区的人类、猪和其他哺乳动物发生病毒性脑炎。CRISPR 筛选、DNA 测序和 RNA 干扰等遗传筛选工具极大地提高了我们对 JEV 复制及其潜在抗病毒方法的认识。然而，与 JEV 复制相关的外显子和内含子突变的信息仍然很少。CRISPR-Cas9 介导的胞嘧啶碱基编辑可以有效地在活细胞的基因组中产生 C:G 到 T:A 的转换。碱基编辑的一个有趣应用是筛选尚未在猪中实现的基因功能的关键变体。在这里，我们说明 CRISPR-Cas9 介导的胞嘧啶碱基编辑器，称为 AncBE4max，可以用于 calreticulin()变体的功能分析。我们针对 所有外显子和内含子进行了 CRISPR-Cas9 介导的胞嘧啶碱基编辑筛选，以鉴定参与 JEV 复制的无功能变体。我们出人意料地发现，两个富集的 sgRNA 靶向 基因内含子 2 中的相同位点。我们发现，将 基因内含子 2 中的四个连续 G 碱基突变为四个 A 碱基，可显著抑制 JEV 复制。因此，我们在猪中建立了 CRISPR-Cas9 介导的胞嘧啶碱基编辑点突变筛选技术。我们的结果表明，CRISPR 介导的碱基编辑是鉴定 基因编码和非编码区域中变体的抗病毒功能的强大工具。
