Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements

Molly Gasperini, Gregory M. Findlay, Aaron McKenna, Jennifer H. Milbank, Choli Lee, Melissa D. Zhang, Darren A. Cusanovich, Jay Shendure
doi: https://doi.org/10.1101/092445
Molly Gasperini
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: shendure@uw.edu gasperim@uw.edu
Gregory M. Findlay
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Aaron McKenna
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jennifer H. Milbank
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Choli Lee
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Melissa D. Zhang
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Darren A. Cusanovich
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jay Shendure
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
2Howard Hughes Medical Institute, Seattle, WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: shendure@uw.edu gasperim@uw.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

Abstract

The extent to which distal non-coding mutations contribute to Mendelian disease remains a major unknown in human genetics. Given that a gene’s in vivo function can be appropriately modeled in vitro, CRISPR/Cas9 genome editing enables the large-scale perturbation of distal non-coding regions to identify functional elements in their native context. However, early attempts at such screens have relied on one individual guide RNA (gRNA) per cell, resulting in sparse mutagenesis with minimal redundancy across regions of interest. To address this, we developed a system that uses pairs of gRNAs to program thousands of kilobase-scale deletions that scan across a targeted region in a tiling fashion (“ScanDel”). As a proof-of-concept, we applied ScanDel to program 4,342 overlapping 1- and 2- kilobase (Kb) deletions that tile a 206 Kb region centered on HPRT1, the gene underlying Lesch-Nyhan syndrome, with median 27-fold redundancy per base. Programmed deletions were functionally assayed by selecting for loss of HPRT1 function with 6-thioguanine. HPRT1 exons served as positive controls, and all were successfully identified as functionally critical by the screen. Remarkably, HPRT1 function appeared robust to deletion of any intergenic or deeply intronic non-coding region across the 206 Kb locus, indicating that proximal regulatory sequences are sufficient for its expression. A sparser mutagenesis screen of the same 206 Kb with individual gRNAs also failed to identify critical distal regulatory elements. Although our screen did find programmed deletions and individual gRNAs with putative functional consequences that targeted exon-proximal non-coding sequences (e.g. the promoter), long-read sequencing revealed that this signal was driven almost entirely by rare, unexpected deletions that extended into exonic sequence. These targeted validation experiments defined a small region surrounding the transcriptional start site as the only non-coding sequence essential to HPRT1 function. Overall, our results suggest that distal regulatory elements are not critical for HPRT1 expression, and underscore the necessity of comprehensive edited-locus genotyping for validating the results of CRISPR screens. The application of ScanDel to additional loci will enable more insight into the extent to which the disruption of distal non-coding elements contributes to Mendelian diseases. In addition, dense, redundant, large-scale deletion scanning with gRNA pairs will facilitate a deeper understanding of endogenous gene regulation in the human genome.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted December 08, 2016.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements
Molly Gasperini, Gregory M. Findlay, Aaron McKenna, Jennifer H. Milbank, Choli Lee, Melissa D. Zhang, Darren A. Cusanovich, Jay Shendure
bioRxiv 092445; doi: https://doi.org/10.1101/092445
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools
Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements
Molly Gasperini, Gregory M. Findlay, Aaron McKenna, Jennifer H. Milbank, Choli Lee, Melissa D. Zhang, Darren A. Cusanovich, Jay Shendure
bioRxiv 092445; doi: https://doi.org/10.1101/092445

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Genomics
Subject Areas
All Articles
  • Animal Behavior and Cognition (4379)
  • Biochemistry (9571)
  • Bioengineering (7082)
  • Bioinformatics (24824)
  • Biophysics (12595)
  • Cancer Biology (9944)
  • Cell Biology (14333)
  • Clinical Trials (138)
  • Developmental Biology (7942)
  • Ecology (12092)
  • Epidemiology (2067)
  • Evolutionary Biology (15979)
  • Genetics (10915)
  • Genomics (14728)
  • Immunology (9859)
  • Microbiology (23636)
  • Molecular Biology (9472)
  • Neuroscience (50818)
  • Paleontology (369)
  • Pathology (1538)
  • Pharmacology and Toxicology (2677)
  • Physiology (4006)
  • Plant Biology (8651)
  • Scientific Communication and Education (1508)
  • Synthetic Biology (2389)
  • Systems Biology (6420)
  • Zoology (1345)