New York Genome Center, New York, NY, USA.
Department of Systems Biology, Columbia University, New York, NY, USA.
Genome Med. 2020 Sep 11;12(1):79. doi: 10.1186/s13073-020-00777-8.
We present an assay to experimentally test the regulatory effects of genetic variants within transcripts using CRISPR/Cas9 followed by targeted sequencing. We applied the assay to 32 premature stop-gained variants across the genome and in two Mendelian disease genes, 33 putative causal variants of eQTLs, and 62 control variants in HEK293T cells, replicating a subset of variants in HeLa cells. We detected significant effects in the expected direction (in 60% of variants), demonstrating the ability of the assay to capture regulatory effects of eQTL variants and nonsense-mediated decay triggered by premature stop-gained variants. The results suggest a utility for validating transcript-level effects of genetic variants.
我们提出了一种使用 CRISPR/Cas9 进行实验测试转录本中遗传变异的调控效应，然后进行靶向测序的方法。我们将该方法应用于全基因组中的 32 个提前终止获得变异，以及两个孟德尔疾病基因、33 个推定的 eQTL 因果变异，和 62 个 HEK293T 细胞中的对照变异，在 HeLa 细胞中复制了一部分变异。我们在预期的方向上检测到了显著的效应（在 60%的变异中），证明了该方法能够捕捉到 eQTL 变异的调控效应和由提前终止获得变异引发的无意义介导的衰变。这些结果表明该方法可用于验证遗传变异在转录水平上的影响。
