CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions
CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions
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DOI：
10.1016/j.ajhg.2017.06.010
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发表时间：
2017-08-03
影响因子：
9.8
通讯作者：
Shendure, Jay
中科院分区：
文献类型：
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作者：
Gasperini, Molly;Findlay, Gregory M.;Shendure, Jay
非编码突变对孟德尔病的影响程度在人类遗传学中是一个主要未知数。与此相关的是，绝大多数候选监管要素尚未得到功能验证。在这里，我们描述了一个基于 CRISPR 的系统，该系统使用成对的引导 RNA (gRNA) 来编程数千个千碱基级的删除，以平铺方式深度扫描目标区域 (“ScanDel”)。我们将 ScanDel 应用于 HPRT1，HPRT1 是 Lesch-Nyhan 综合征（一种 X 连锁隐性遗传病）背后的管家基因。总共，我们编程了 4,342 个重叠的 1 和 2 kb 删除，平铺了 206 个kb 以 HPRT1 为中心（包括 87 kb 上游和 79 kb 下游），每个碱基冗余度中位数为 27 倍。正如预期的那样，通过使用 6-硫鸟嘌呤选择 HPRT 功能的缺失，对 HPRT1 外显子进行测序，证实需要所有 HPRT1 外显子。尽管我们的筛选确实确定了外显子近端非编码序列（例如启动子）的破坏是功能上的后果，但长读长测序表明该信号是由延伸到外显子的罕见、不精确的缺失驱动的，我们的结果表明 HPRT1 表达不需要单一的远端调控元件，并且远端突变不太可能对 Lesch-Nyhan 综合征负担产生重大影响。位点，同时还有助于更深入地了解内源基因调控。
The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel''). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation.