Protacio Reine U, Malone Emory G, Wahls Wayne P
Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States.
MicroPubl Biol. 2024 Jul 25;2024. doi: 10.17912/micropub.biology.001248. eCollection 2024.
Discrete DNA sites position meiotic recombination at hotspots. We sought to create four different, 15 bp long, candidate regulatory DNA sites within the 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, , has two substitutions (C379T, C380A) that create a stop codon, rendering strains unable to grow without uracil.
离散的DNA位点将减数分裂重组定位在热点区域。我们试图在报告基因内创建四个不同的、长度为15 bp的候选调控DNA位点。每次尝试都使用了经裂殖酵母优化的CRISPR系统（SpEDIT）、最佳引导RNA以及四个具有10至15 bp替换的同源重组模板之一。值得注意的是，每个分析的尿嘧啶转化体在DSB附近（距离5 - 6 bp）都有模板导向的、使PAM失活的bp替换，但在远距离（42 - 56 bp）没有产生DNA位点的替换。一个意外发现的新等位基因有两个替换（C379T、C380A），产生了一个终止密码子，使得菌株在没有尿嘧啶的情况下无法生长。
