PDF(Pubmed)
Abstract：
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 (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR 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 CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR 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 CALR gene.
摘要：
日本脑炎病毒（JEV）是一种蚊子传播的黄病毒，可引起人类的病毒性脑炎，亚洲和西太平洋的猪和其他哺乳动物。基因筛选工具，如CRISPR筛选，DNA测序和RNA干扰极大地改善了我们对JEV复制及其潜在抗病毒方法的理解。然而,关于与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基因组中产生C:G到T:A转换。碱基编辑的一个有趣的应用是筛选基因功能的关键变体，这在猪中尚未实现。这里,我们说明了CRISPR-Cas9介导的胞嘧啶碱基编辑器，被称为AncBE4max，可用于钙网蛋白（CALR）变体的功能分析。我们针对CALR的所有外显子和内含子使用457个单指导RNA（sgRNA）进行了CRISPR-Cas9介导的胞嘧啶碱基编辑筛选，以鉴定参与JEV复制的功能丧失变体。我们意外地发现两个富集的sgRNA靶向CALR基因内含子2中的相同位点。我们发现，将CALR基因内含子2中的四个连续G碱基突变为四个A碱基可显着抑制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CALR基因编码区和非编码区变体抗病毒功能的有力工具。
