In vivo  profiling of metastatic double knockouts through CRISPR-Cpf1 screens

Chow, Ryan D; Wang, Guangchuan; Ye, Lupeng; Codina, Adan; Kim, Hyunu Ray; Shen, Li; Dong, Matthew B; Errami, Youssef; Chen, Sidi

doi:10.1038/s41592-019-0371-5

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In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens

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Author(s): Ryan D. Chow ¹ ^, ² ^, ³ ^, ⁴ , Guangchuan Wang ¹ ^, ² ^, ³ , Lupeng Ye ¹ ^, ² ^, ³ , Adan Codina ¹ ^, ² ^, ³ ^, ⁵ , Hyunu Ray Kim ¹ ^, ² ^, ³ , Li Shen ¹ ^, ² ^, ³ , Matthew B. Dong ¹ ^, ² ^, ³ ^, ⁴ ^, ⁶ , Youssef Errami ¹ ^, ² ^, ³ , Sidi Chen ¹ ^, ² ^, ³ ^, ⁴ ^, ⁵ ^, ⁶ ^, ⁷ ^, ⁸ ^, ^*

Publication date (Electronic): 08 April 2019

Journal: Nature methods

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Abstract

The genetic interactions influencing metastatic potential have been challenging to investigate systematically. Here we developed MCAP (massively parallel CRISPR-Cpf1/Cas12a crRNA array profiling), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library of 11,934 arrays targeting 325 pairwise combinations of genes implicated in metastasis. By assessing the metastatic potential of the double knockouts in mice, we unveiled a quantitative landscape of genetic interactions driving metastasis.

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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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In vivo high-throughput profiling of CRISPR–Cpf1 activity

Hui Kim, Myungjae Song, Jinu Lee … (2016)

A lentiviral library expressing Cpf1 guide RNAs and containing target sequences allows high-throughput profiling of highly active guide RNAs and is the basis for cindel, a webtool to predict the activity at any given target sequence.

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Dicer1 functions as a haploinsufficient tumor suppressor.

Madhu S. Kumar, Ryan Pester, Cindy Chen … (2009)

While the global down-regulation of microRNAs (miRNAs) is a common feature of human tumors, its genetic basis is largely undefined. To explore this question, we analyzed the consequences of conditional Dicer1 mutation (Dicer1 "floxed" or Dicer1(fl)) on several mouse models of cancer. Here we show Dicer1 functions as a haploinsufficient tumor suppressor gene. Deletion of a single copy of Dicer1 in tumors from Dicer1(fl/+) animals led to reduced survival compared with controls. These tumors exhibited impaired miRNA processing but failed to lose the wild-type Dicer1 allele. Moreover, tumors from Dicer1(fl/fl) animals always maintained one functional Dicer1 allele. Consistent with selection against full loss of Dicer1 expression, enforced Dicer1 deletion caused inhibition of tumorigenesis. Analysis of human cancer genome copy number data reveals frequent deletion of DICER1. Importantly, however, the gene has not been reported to undergo homozygous deletion, suggesting that DICER1 is haploinsufficient in human cancer. These findings suggest Dicer1 may be an important haploinsufficient tumor suppressor gene and, furthermore, that other factors controlling miRNA biogenesis may also function in this manner.

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Author and article information

Journal

Journal ID (nlm-journal-id): 101215604

Journal ID (pubmed-jr-id): 32338

Journal ID (nlm-ta): Nat Methods

Journal ID (iso-abbrev): Nat. Methods

Title: Nature methods

ISSN (Print): 1548-7091

ISSN (Electronic): 1548-7105

Publication date Nihms-submitted: 21 March 2019

Publication date (Electronic): 08 April 2019

Publication date (Print): May 2019

Publication date PMC-release: 08 October 2019

Volume: 16

Issue: 5

Pages: 405-408

Affiliations

[1 ]Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA

[2 ]System Biology Institute, Integrated Science & Technology Center, Yale University, West Haven, Connecticut, USA

[3 ]Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, Connecticut, USA

[4 ]Yale M.D.-Ph.D. Program, New Haven, Connecticut, USA

[5 ]Yale Combined Program in the Biological and Biomedical Sciences, New Haven, Connecticut, USA

[6 ]Yale Immunobiology Program, The Anlyan Center for Medical Research & Education, New Haven, Connecticut, USA

[7 ]Yale Comprehensive Cancer Center, New Haven, Connecticut, USA

[8 ]Yale Stem Cell Center, New Haven, Connecticut, USA

Author notes

Contributions

R.D.C. designed the study, performed experiments, developed statistical algorithms and computational pipelines, and analyzed the data. G.W. and L.Y. performed molecular, cellular, animal experiments and MCAP readout. A.C. optimized in vivo screens and assisted with validation experiments. H.R.K. and L.S. assisted with cloning and validation experiments. M.B.D. assisted with flow cytometry experiments. Y.E. assisted with in vitro assays. S.C. conceived the study, provided conceptual advice, secured funding and supervised the work. R.D.C. and S.C. wrote the manuscript.

^† Co-first authors

[* ]Correspondence: SC ( sidi.chen@ 123456yale.edu ), +1-203-737-3825 (office), +1-203-737-4952 (lab)

Article

Manuscript ID: NIHMS1523232

DOI: 10.1038/s41592-019-0371-5

PMC ID: 6592845

PubMed ID: 30962622

SO-VID: 82b40885-c93e-4cc7-b739-5fa6bc8c320a

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In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens

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Genome Engineering using CRISPR

Most cited references 12

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

In vivo high-throughput profiling of CRISPR–Cpf1 activity

Dicer1 functions as a haploinsufficient tumor suppressor.

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