FlashFry: a fast and flexible tool for large-scale CRISPR target design

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Abstract

Background

Genome-wide knockout studies, noncoding deletion scans, and other large-scale studies require a simple and lightweight framework that can quickly discover and score thousands of candidate CRISPR guides targeting an arbitrary DNA sequence. While several CRISPR web applications exist, there is a need for a high-throughput tool to rapidly discover and process hundreds of thousands of CRISPR targets.

Results

Here, we introduce FlashFry, a fast and flexible command-line tool for characterizing large numbers of CRISPR target sequences. With FlashFry, users can specify an unconstrained number of mismatches to putative off-targets, richly annotate discovered sites, and tag potential guides with commonly used on-target and off-target scoring metrics. FlashFry runs at speeds comparable to commonly used genome-wide sequence aligners, and output is provided as an easy-to-manipulate text file.

Conclusions

FlashFry is a fast and convenient command-line tool to discover and score CRISPR targets within large DNA sequences.

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Most cited references 4

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Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis.

Sidi Chen, Neville E. Sanjana, Kaijie Zheng … (2015)

Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.

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A guided tour to approximate string matching

Gonzalo Navarro (2001)

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CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions

Choli Lee, Melissa D. Zhang, Darren Cusanovich … (2017)

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.

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Author and article information

Contributors

Aaron McKenna:

ORCID: http://orcid.org/0000-0001-8277-6512

aaronmck@uw.edu

Journal

Journal ID (nlm-ta): BMC Biol

Journal ID (iso-abbrev): BMC Biol

Title: BMC Biology

Publisher: BioMed Central (London )

ISSN (Electronic): 1741-7007

Publication date (Electronic): 5 July 2018

Publication date PMC-release: 5 July 2018

Publication date Collection: 2018

Volume: 16

Electronic Location Identifier: 74

Affiliations

[1 ]ISNI 0000000122986657, GRID grid.34477.33, Department of Genome Sciences, , University of Washington, ; Seattle, WA USA

[2 ]ISNI 0000 0001 2167 1581, GRID grid.413575.1, Howard Hughes Medical Institute, ; Seattle, WA USA

Author information

Aaron McKenna http://orcid.org/0000-0001-8277-6512

Article

Publisher ID: 545

DOI: 10.1186/s12915-018-0545-0

PMC ID: 6033233

PubMed ID: 29976198

SO-VID: eb1ba5d2-ab62-4db1-ad41-f602c365e0c7

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 20 May 2018

Date accepted : 22 June 2018

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000051, National Human Genome Research Institute;

Award ID: R01HG006283

Award Recipient : Jay Shendure

Funded by: FundRef http://dx.doi.org/10.13039/100000050, National Heart, Lung, and Blood Institute;

Award ID: T32HL007312

Award Recipient : Aaron McKenna

Custom metadata

ScienceOpen disciplines: Life sciences

Keywords: crispr,guide library,cas9,cpf1,off-target,on-target,deletion scan

Data availability:

ScienceOpen disciplines: Life sciences

Keywords: crispr, guide library, cas9, cpf1, off-target, on-target, deletion scan

FlashFry: a fast and flexible tool for large-scale CRISPR target design

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Abstract

Background

Results

Conclusions

Related collections

CRISPR/Cas9 editing in human blood

Most cited references 4

Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis.

A guided tour to approximate string matching

CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions

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