CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in <i xmlns='http://www.wiley.com/namespaces/wiley'>Yarrowia lipolyticaYarrowia lipolytica

Schwartz Cory; Frogue Keith; Ramesh Adithya; Misa Joshua; Wheeldon Ian

摘要

Abstract <section xml:id="bit26404-sec-0001" numbered="no"> > In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical‐producing yeast Yarrowia lipolytica . By using a multiplexed sgRNA targeting strategy, we demonstrate efficient repression of eight out of nine targeted genes to enhance HR. Strains with nonhomologous end‐joining repressed were shown to have increased rates of HR when transformed with a linear DNA fragment with homology to a genomic locus. With multiplexed targeting of KU70 and KU80 , and enhanced repression with Mxi1 fused to deactivated Cas9 (dCas9), rates of HR as high as 90% were achieved. The developed CRISPRi system enables enhanced HR in Y. lipolytica without permanent genetic knockouts and promises to be a potent tool for other metabolic engineering, synthetic biology, and functional genomics studies. </section> </abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> <div class="translation abstracttxt"> <span class="zhankaihshouqi fivelineshidden" id="abstract"> <span>机译：</span><Abstract XMLNS =“http://www.wiley.com/namespaces/wiley”type =“main”xml：lang =“en”> <title type =“main”>抽象</ title> <section XML：ID =“Bit26404-SEC-0001”编号=“否”> >在许多生物技术重要性的生物中，精确的基因组编辑受到固有的低同源重组（HR）效率的限制。存在许多策略以提高该天然DNA修复途径的有效性;然而，大多数策略依赖于永久性致力于竞争的修复途径，从而降低了有机体的维修地发生了自然发生的双链断裂的能力。在这里，我们描述了在油化学生产酵母中的基因抑制的CRISPR干扰（CRISPRI）系统 Yarrowia Lipolytica </ i>。通过使用多路复用的SGRNA靶向策略，我们证明了八个患有九个靶向基因的八个以增强人力资源的抑制。当用与基因组基因座同源性转化时，具有非分h原始终端接合抑制的菌株具有增加的HR速率。随着 Ku70 </ i>和 Ku80 </ i>的多路复用靶向，并用MXI1融合到去激活的CAS9（DCAS9）的增强抑制，实现了高达90％的HR的速率。发达的CRISPRI系统可以在 y中获得增强的HR。 Lipolytica </ i>没有永久性遗传淘汰，并承诺成为其他代谢工程，合成生物学和功能基因组学研究的有效工具。 </ p> </ section> </ abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> </div> <div class="record"> <h2 class="all_title" id="enpatent33" >著录项</h2> <ul> <li> <span class="lefttit">来源</span> <div style="width: 86%;vertical-align: text-top;display: inline-block;"> <a href='/journal-foreign-15017/'>《Biotechnology and Bioengineering》</a> <b style="margin: 0 2px;">|</b><span>2017年第12期</span><b style="margin: 0 2px;">|</b><span>共11页</span> </div> </li> <li> <div class="author"> <span class="lefttit">作者</span> <p id="fAuthorthree" class="threelineshidden zhankaihshouqi"> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Schwartz Cory&option=202" target="_blank" rel="nofollow">Schwartz Cory;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Frogue Keith&option=202" target="_blank" rel="nofollow">Frogue Keith;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Ramesh Adithya&option=202" target="_blank" rel="nofollow">Ramesh Adithya;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Misa Joshua&option=202" target="_blank" rel="nofollow">Misa Joshua;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Wheeldon Ian&option=202" target="_blank" rel="nofollow">Wheeldon Ian;</a> </p> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zkzz" style="display: none;">展开▼</span> </div> </li> <li> <div style="display: flex;"> <span class="lefttit">作者单位</span> <div style="position: relative;margin-left: 3px;max-width: 639px;"> <div class="threelineshidden zhankaihshouqi" id="fOrgthree"> <p>Chemical and Environmental EngineeringUniversity of California RiversideRiverside California;</p> <p>Chemical and Environmental EngineeringUniversity of California RiversideRiverside California;</p> <p>Chemical and Environmental EngineeringUniversity of California RiversideRiverside California;</p> <p>Chemical and Environmental EngineeringUniversity of California RiversideRiverside California;</p> <p>Chemical and Environmental EngineeringUniversity of California RiversideRiverside California;</p> </div> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zhdw" style="display: none;">展开▼</span> </div> </div> </li> <li > <span class="lefttit">收录信息</span> <span style="width: 86%;vertical-align: text-top;display: inline-block;"></span> </li> <li> <span class="lefttit">原文格式</span> <span>PDF</span> </li> <li> <span class="lefttit">正文语种</span> <span>eng</span> </li> <li> <span class="lefttit">中图分类</span> <span><a href="https://www.zhangqiaokeyan.com/clc/180.html" title="生物工程学（生物技术）">生物工程学（生物技术）;</a></span> </li> <li class="antistop"> <span class="lefttit">关键词</span> <p style="width: 86%;vertical-align: text-top;"> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=CRISPR interference&option=203" rel="nofollow">CRISPR interference;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=DNA repair&option=203" rel="nofollow">DNA repair;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=gene repression&option=203" rel="nofollow">gene repression;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=genome engineering&option=203" rel="nofollow">genome engineering;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=non‐conventional yeast&option=203" rel="nofollow">non‐conventional yeast;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=transcriptional regulation&option=203" rel="nofollow">transcriptional regulation;</a> </p> <div class="translation"> 机译：CRISPR干扰;DNA修复;基因抑制;基因组工程;非常规酵母;转录调节; </div> </li> </ul> </div> </div> <div class="literature cardcommon"> <div class="similarity "> <h3 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CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in Yarrowia lipolyticaYarrowia lipolytica

摘要