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首页> 外文会议>AIChE Annual Meeting >Homology-lntegrated CRISPR-Cas (HI-CRISPR) System for One-Step Multigene Disruption in Saccharomyces cerevisiae
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Homology-lntegrated CRISPR-Cas (HI-CRISPR) System for One-Step Multigene Disruption in Saccharomyces cerevisiae

机译：同源性-LN的CRAP-CAS（HI-CRISPR）Saccharomyces酿酒酵母中的一步多岛破坏系统

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摘要

One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.
机译：模型生物体酿酒酵母中的一步多基因破坏是基本和应用研究的一个非常有用的工具，但它仍然是一个挑战。在这里，我们报告了基于II型聚集的快速，有效和潜在可扩展的策略，该型群体定期间隙的短语重复（CRISPR）-CRISPR相关蛋白（CAS）系统，以在S.Cerevisiae中同时产生多种基因破坏。在由多个供体和引导序列对组成的CRISPR阵列中，将100bp DSDNA诱变同源重组供体在两种直接重复之间插入两种直接重复之间。携带ICAS9的超高拷贝数质粒，野生型CAS9的变体，反式编码的RNA（TRACRRNA）和同源集成CRRNA盒的设计，以大大提高基因破坏效率。作为概念证明，三个基因，CAN1，ADE2和LYP1在4天内同时破坏，效率范围为27％至87％。参与人工氢化可的酮的另外三种基因，ATF2，GCY1和YPR1在6天内同时破坏100％效率。这种同源集成的CRISPR（HI-CRISPR）策略代表了一种强大的工具，用于创造具有多种基因敲门的酵母菌株。

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