Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast.
Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast.
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DOI：
10.1016/j.crmeth.2023.100464
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发表时间：
2023-05-22
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影响因子：
--
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在酵母中合理构建多基因过程的一个主要挑战是将所有个体编辑组合到同一菌株中的组合学。在这里，我们提出了一种精确的多位点基因组编辑方法，该方法使用CRISPR-Cas9组合了所有编辑而没有选择标记。我们展示了一种高效的基因驱动，通过整合CRISPR-Cas9介导的双链断裂（DSB）生成和同源定向重组与酵母性别分类来选择性消除特定位点。该方法能够实现遗传工程化基因座的无标记富集和重组（MERGE）。我们表明，MERGE转换单一的异源基因座纯合基因座在100%的效率，独立的染色体位置。此外，MERGE在转换和组合多个基因座方面同样有效，从而鉴定相容的基因型。最后，我们通过将真菌类胡萝卜素生物合成途径和大部分人类α-蛋白酶体核心工程化到酵母中来建立MERGE能力。因此，MERGE为酵母中可扩展的组合基因组编辑奠定了基础。Cas9诱导的基因驱动有效地将杂合转化为纯合酵母基因座酵母交配和Cas9选择将两个或更多个单独的编辑组合到单个菌株中该方法能够实现无标记富集，基因工程基因座的重组MERGE揭示了在酵母中人源化α-蛋白酶体核心亚基的适合度驱动的路径工程化酵母中的整个生物过程，控制表达和基因组背景，由于使用线性外源修复模板的低效同源定向修复（HDR），通常导致不可定量的读数，因此具有挑战性。因此，不能获得编辑可归因于低效HDR或不可行基因型。因此，实现多重和可扩展的基因组编辑需要兼容的技术。我们开发了一种基于CRISPR-Cas9的组合基因组编辑方法（MERGE），该方法有助于所有个体基因编辑的适应度驱动组合，消除了对选择标记的需要，同时使用菌落形成单位的定量读数。Abdullah等人使用酵母交配和CRISPR-Cas9选择将联合收割机许多单独的遗传编辑组合成单个菌株。所述方法促进遗传工程化基因座的无标记富集和重组（MERGE）。MERGE通过探索适应度景观来设计酵母中的整个遗传系统。
A major challenge to rationally building multi-gene processes in yeast arises due to the combinatorics of combining all of the individual edits into the same strain. Here, we present a precise and multi-site genome editing approach that combines all edits without selection markers using CRISPR-Cas9. We demonstrate a highly efficient gene drive that selectively eliminates specific loci by integrating CRISPR-Cas9-mediated double-strand break (DSB) generation and homology-directed recombination with yeast sexual assortment. The method enables marker-less enrichment and recombination of genetically engineered loci (MERGE). We show that MERGE converts single heterologous loci to homozygous loci at ∼100% efficiency, independent of chromosomal location. Furthermore, MERGE is equally efficient at converting and combining multiple loci, thus identifying compatible genotypes. Finally, we establish MERGE proficiency by engineering a fungal carotenoid biosynthesis pathway and most of the human α-proteasome core into yeast. Therefore, MERGE lays the foundation for scalable, combinatorial genome editing in yeast. Cas9-induced gene drive efficiently converts heterozygous to homozygous yeast locus Yeast mating and Cas9 selection combines two or more separate edits into a single strain The method enables marker-less enrichment, recombination of genetically engineered loci MERGE reveals a fitness-driven path to humanize α-proteasome core subunits in yeast Engineering entire biological processes in yeast, controlled for expression and genomic context, is challenging due to inefficient homology-directed repair (HDR) using linear exogenous repair templates, often resulting in non-quantifiable readouts. Thus, an inability to obtain the edit can be attributed to inefficient HDR or inviable genotype. Therefore, accomplishing multiplexed and scalable genomic editing necessitates compatible technology. We developed a CRISPR-Cas9-based combinatorial genome editing method (MERGE) that facilitates a fitness-driven combination of all individual genetic edits, eliminating the need for selection markers while using a quantitative readout of colony-forming units. Abdullah et al. use yeast mating and CRISPR-Cas9 selection to combine many individual genetic edits into a single strain. The method facilitates marker-less enrichment and recombination of genetically engineered loci (MERGE). MERGE engineers the entire genetic systems in yeast by exploring the fitness landscape.
影响因子：
13.5
作者：
Smith, Andrew M.;Ammar, Ron;Nislow, Corey;Giaever, Guri
通讯作者：
Giaever, Guri
影响因子：
7
作者：
Suzuki Y;Assad-Garcia N;Kostylev M;Noskov VN;Wise KS;Karas BJ;Stam J;Montague MG;Hanly TJ;Enriquez NJ;Ramon A;Goldgof GM;Richter RA;Vashee S;Chuang RY;Winzeler EA;Hutchison CA 3rd;Gibson DG;Smith HO;Glass JI;Venter JC
通讯作者：
Venter JC