{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T13:08:38Z","timestamp":1787317718656,"version":"build-2736575974"},"reference-count":40,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2023,8,28]],"date-time":"2023-08-28T00:00:00Z","timestamp":1693180800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Natural Science Foundation of China","award":["32072685"],"award-info":[{"award-number":["32072685"]}]},{"name":"Natural Science Foundation of China","award":["2021YFA0805903-3"],"award-info":[{"award-number":["2021YFA0805903-3"]}]},{"name":"Natural Science Foundation of China","award":["2020ABA016"],"award-info":[{"award-number":["2020ABA016"]}]},{"name":"National Key R&amp;D Program of China","award":["32072685"],"award-info":[{"award-number":["32072685"]}]},{"name":"National Key R&amp;D Program of China","award":["2021YFA0805903-3"],"award-info":[{"award-number":["2021YFA0805903-3"]}]},{"name":"National Key R&amp;D Program of China","award":["2020ABA016"],"award-info":[{"award-number":["2020ABA016"]}]},{"name":"Major Science and Technology Projects in Hubei Province","award":["32072685"],"award-info":[{"award-number":["32072685"]}]},{"name":"Major Science and Technology Projects in Hubei Province","award":["2021YFA0805903-3"],"award-info":[{"award-number":["2021YFA0805903-3"]}]},{"name":"Major Science and Technology Projects in Hubei Province","award":["2020ABA016"],"award-info":[{"award-number":["2020ABA016"]}]},{"name":"Supported by Hubei Provincial Natural Science Foundation of China","award":["32072685"],"award-info":[{"award-number":["32072685"]}]},{"name":"Supported by Hubei Provincial Natural Science Foundation of China","award":["2021YFA0805903-3"],"award-info":[{"award-number":["2021YFA0805903-3"]}]},{"name":"Supported by Hubei Provincial Natural Science Foundation of China","award":["2020ABA016"],"award-info":[{"award-number":["2020ABA016"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/ijms241713331","type":"journal-article","created":{"date-parts":[[2023,8,28]],"date-time":"2023-08-28T05:46:47Z","timestamp":1693201607000},"page":"13331","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["CRISPR-Cas9-Mediated Cytosine Base Editing Screen for the Functional Assessment of CALR Intron Variants in Japanese Encephalitis Virus Replication"],"prefix":"10.3390","volume":"24","author":[{"given":"Youcai","family":"Xiong","sequence":"first","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoning","family":"Xi","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Xiang","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sheng","family":"Li","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hailong","family":"Liu","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yinyu","family":"Su","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruigao","family":"He","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chong","family":"Xiong","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bingrong","family":"Xu","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyi","family":"Wang","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liangliang","family":"Fu","sequence":"additional","affiliation":[{"name":"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"},{"name":"The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changzhi","family":"Zhao","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaosong","family":"Han","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyun","family":"Li","sequence":"additional","affiliation":[{"name":"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"},{"name":"The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8301-877X","authenticated-orcid":false,"given":"Shengsong","family":"Xie","sequence":"additional","affiliation":[{"name":"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"},{"name":"The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinxue","family":"Ruan","sequence":"additional","affiliation":[{"name":"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"},{"name":"The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"698","DOI":"10.4269\/ajtmh.1959.8.698","article-title":"Ecologic studies of Japanese encephalitis virus in Japan. 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