Saccharomyces cerevisiae is a genetically facile organism, yet multiple CRISPR/Cas9 techniques are widely used to edit its genome more efficiently and cost effectively than conventional methods. The absence of selective markers makes CRISPR/Cas9 editing particularly useful when making mutations within genes or regulatory sequences. Heterozygous mutations within genes frequently arise in the winners of evolution experiments. The genetic dissection of heterozygous alleles can be important to understanding gene structure and function. Unfortunately, the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by standard CRISPR/Cas9 technique impossible. To be able to quickly and reliably determine the individual phenotypes of the thousands of heterozygous mutations that can occur during directed evolutions is of particular interest to industrial strain improvement research. In this report, we describe a CRISPR/Cas9 method that introduces specific heterozygous mutations into the S. cerevisiae genome. This method relies upon creating silent point mutations in the protospacer adjacent motif site or removing the protospacer adjacent motif site entirely to stop the multiple rounds of genome editing that prevent heterozygous alleles from being generated. This technique should be able to create heterozygous alleles in other diploid yeasts and different allelic copy numbers in polyploid cells.

中文翻译：

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CRISPR / Cas9方法在酿酒酵母中产生杂合等位基因。

酿酒酵母（Saccharomyces cerevisiae）是一种遗传上易变的生物，但是与传统方法相比，多种CRISPR / Cas9技术被广泛用于更有效和经济地编辑其基因组。当在基因或调控序列内进行突变时，缺少选择性标记使CRISPR / Cas9编辑特别有用。基因内的杂合突变经常发生在进化实验的赢家中。杂合等位基因的遗传解剖对于理解基因结构和功能可能很重要。不幸的是，基因组切割和修复的高效率使得不可能通过标准CRISPR / Cas9技术引入杂合等位基因。为了能够快速，可靠地确定在定向进化过程中可能发生的数千个杂合突变的个体表型，对工业菌株改良研究尤为重要。在此报告中，我们描述了一种CRISPR / Cas9方法，该方法将特定的杂合突变引入酿酒酵母基因组中。该方法依赖于在原间隔物相邻基序位点中产生沉默点突变或完全去除原间隔物相邻基序位点以停止多轮基因组编辑，从而防止杂合等位基因的产生。该技术应该能够在其他二倍体酵母中创建杂合等位基因，并在多倍体细胞中创建不同的等位基因拷贝数。我们描述了一种将特定的杂合突变引入酿酒酵母基因组的CRISPR / Cas9方法。该方法依赖于在原间隔物相邻基序位点中产生沉默点突变或完全去除原间隔物相邻基序位点以停止多轮基因组编辑，从而防止杂合等位基因的产生。该技术应该能够在其他二倍体酵母中创建杂合等位基因，并在多倍体细胞中创建不同的等位基因拷贝数。我们描述了一种将特定的杂合突变引入酿酒酵母基因组的CRISPR / Cas9方法。该方法依赖于在原间隔物相邻基序位点中产生沉默点突变或完全去除原间隔物相邻基序位点以停止多轮基因组编辑，从而防止杂合等位基因的产生。该技术应该能够在其他二倍体酵母中创建杂合等位基因，并在多倍体细胞中创建不同的等位基因拷贝数。