{"title":"距离依赖性效应影响了球囊酵母菌（Schizosaccharomyces pombe）中 CRISPR/Cas9 介导的基因组编辑的效率，并产生了不需要的等位基因。","authors":"Reine U Protacio, Emory G Malone, Wayne P Wahls","doi":"10.17912/micropub.biology.001248","DOIUrl":null,"url":null,"abstract":"<p><p>Discrete DNA sites position meiotic recombination at hotspots. We sought to create four different, 15 bp long, candidate regulatory DNA sites within the <i>ura4</i> reporter gene. Each effort employed a fission yeast-optimized CRISPR system (SpEDIT), optimal guide RNA, and one of four homologous recombination templates with 10 to 15 bp substitutions. Remarkably, every Ura <sup>-</sup> transformant analyzed had template-directed, PAM-disabling bp substitutions near (5-6 bp away from) the DSB but no DNA site-generating substitutions at distance (42-56 bp). An unsought novel allele, <i>ura4-P127*</i> , has two substitutions (C379T, C380A) that create a stop codon, rendering strains unable to grow without uracil.</p>","PeriodicalId":74192,"journal":{"name":"microPublication biology","volume":"2024 ","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11310776/pdf/","citationCount":"0","resultStr":"{\"title\":\"Distance-dependent effects on CRISPR/Cas9-mediated genome editing in <i>Schizosaccharomyces pombe</i> compromise efficiency and create unsought alleles.\",\"authors\":\"Reine U Protacio, Emory G Malone, Wayne P Wahls\",\"doi\":\"10.17912/micropub.biology.001248\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Discrete DNA sites position meiotic recombination at hotspots. We sought to create four different, 15 bp long, candidate regulatory DNA sites within the <i>ura4</i> reporter gene. Each effort employed a fission yeast-optimized CRISPR system (SpEDIT), optimal guide RNA, and one of four homologous recombination templates with 10 to 15 bp substitutions. Remarkably, every Ura <sup>-</sup> transformant analyzed had template-directed, PAM-disabling bp substitutions near (5-6 bp away from) the DSB but no DNA site-generating substitutions at distance (42-56 bp). An unsought novel allele, <i>ura4-P127*</i> , has two substitutions (C379T, C380A) that create a stop codon, rendering strains unable to grow without uracil.</p>\",\"PeriodicalId\":74192,\"journal\":{\"name\":\"microPublication biology\",\"volume\":\"2024 \",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-07-25\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11310776/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"microPublication biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.17912/micropub.biology.001248\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"microPublication biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.17912/micropub.biology.001248","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
摘要
离散 DNA 位点将减数分裂重组定位在热点上。我们试图在 ura4 报告基因中创建四个不同的、15 bp 长的候选调控 DNA 位点。每种方法都采用了裂殖酵母优化的 CRISPR 系统（SpEDIT）、最佳引导 RNA 和四种同源重组模板中的一种，并进行了 10 到 15 bp 的置换。值得注意的是，分析的每一个 Ura - 转化子都在 DSB 附近（距离 DSB 5-6 bp）有模板定向、PAM 禁用的 bp 置换，但在距离 DSB 42-56 bp 处没有 DNA 位点产生置换。一个未发现的新等位基因 ura4-P127* 有两个取代（C379T、C380A），产生了一个终止密码子，使菌株在没有尿嘧啶的情况下无法生长。
Distance-dependent effects on CRISPR/Cas9-mediated genome editing in Schizosaccharomyces pombe compromise efficiency and create unsought alleles.
Discrete DNA sites position meiotic recombination at hotspots. We sought to create four different, 15 bp long, candidate regulatory DNA sites within the ura4 reporter gene. Each effort employed a fission yeast-optimized CRISPR system (SpEDIT), optimal guide RNA, and one of four homologous recombination templates with 10 to 15 bp substitutions. Remarkably, every Ura - transformant analyzed had template-directed, PAM-disabling bp substitutions near (5-6 bp away from) the DSB but no DNA site-generating substitutions at distance (42-56 bp). An unsought novel allele, ura4-P127* , has two substitutions (C379T, C380A) that create a stop codon, rendering strains unable to grow without uracil.
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