Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA, USA.
Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA, USA.
Nat Genet. 2018 Apr;50(4):510-514. doi: 10.1038/s41588-018-0087-y. Epub 2018 Apr 9.
Understanding the functional effects of DNA sequence variants is of critical importance for studies of basic biology, evolution, and medical genetics; however, measuring these effects in a high-throughput manner is a major challenge. One promising avenue is precise editing with the CRISPR-Cas9 system, which allows for generation of DNA double-strand breaks (DSBs) at genomic sites matching the targeting sequence of a guide RNA (gRNA). Recent studies have used CRISPR libraries to generate many frameshift mutations genome wide through faulty repair of CRISPR-directed breaks by nonhomologous end joining (NHEJ) . Here, we developed a CRISPR-library-based approach for highly efficient and precise genome-wide variant engineering. We used our method to examine the functional consequences of premature-termination codons (PTCs) at different locations within all annotated essential genes in yeast. We found that most PTCs were highly deleterious unless they occurred close to the 3' end of the gene and did not affect an annotated protein domain. Unexpectedly, we discovered that some putatively essential genes are dispensable, whereas others have large dispensable regions. This approach can be used to profile the effects of large classes of variants in a high-throughput manner.
理解 DNA 序列变异的功能影响对于基础生物学、进化和医学遗传学的研究至关重要；然而，以高通量的方式测量这些影响是一个主要的挑战。一种很有前途的方法是使用 CRISPR-Cas9 系统进行精确编辑，该系统允许在与指导 RNA（gRNA）的靶向序列匹配的基因组位点产生 DNA 双链断裂（DSB）。最近的研究使用 CRISPR 文库通过非同源末端连接（NHEJ）对 CRISPR 指导的断裂进行错误修复，从而在全基因组范围内产生了许多移码突变。在这里，我们开发了一种基于 CRISPR 文库的高效、精确的全基因组变体工程方法。我们使用该方法研究了酵母中所有注释必需基因内不同位置的提前终止密码子（PTC）的功能后果。我们发现，大多数 PTC 是高度有害的，除非它们靠近基因的 3'端，并且不影响注释的蛋白质结构域。出乎意料的是，我们发现一些推测的必需基因是可有可无的，而另一些基因则有很大的可有可无区域。这种方法可以用于高通量地分析大量变异类别的影响。
