High-Throughput Approaches to Pinpoint Function Within the Noncoding Genome

Overview

Journal Mol Cell

Publisher Cell Press

Specialty Cell Biology

Date 2017 Oct 7

PMID 28985510

Citations 39

Authors

Antonino Montalbano

Matthew C Canver

Neville E Sanjana

Affiliations

Soon will be listed here.

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease system is a powerful tool for genome editing, and its simple programmability has enabled high-throughput genetic and epigenetic studies. These high-throughput approaches offer investigators a toolkit for functional interrogation of not only protein-coding genes but also noncoding DNA. Historically, noncoding DNA has lacked the detailed characterization that has been applied to protein-coding genes in large part because there has not been a robust set of methodologies for perturbing these regions. Although the majority of high-throughput CRISPR screens have focused on the coding genome to date, an increasing number of CRISPR screens targeting noncoding genomic regions continue to emerge. Here, we review high-throughput CRISPR-based approaches to uncover and understand functional elements within the noncoding genome and discuss practical aspects of noncoding library design and screen analysis.

Citing Articles

Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing.

Rohm D, Black J, McCutcheon S, Barrera A, Berry S, Morone D Cell Genom. 2025; 5(2):100770.

PMID: 39947136 PMC: 11872474. DOI: 10.1016/j.xgen.2025.100770.

Functional RNA mining using random high-throughput screening.

Liu L, Chen J, Lai S, Zhao X, Yang M, Wu Y Nucleic Acids Res. 2024; 53(2.

PMID: 39673274 PMC: 11754670. DOI: 10.1093/nar/gkae1173.

Targeting DLBCL by mutation-specific disruption of cancer-driving oncogenes.

Heshmatpour N, Kazemi S, Schmidt N, Patnaik S, Korus P, Wilkens B Front Genome Ed. 2024; 6:1427322.

PMID: 39469218 PMC: 11513324. DOI: 10.3389/fgeed.2024.1427322.

Discovering the hidden function in fungal genomes.

Gervais N, Shapiro R Nat Commun. 2024; 15(1):8219.

PMID: 39300175 PMC: 11413187. DOI: 10.1038/s41467-024-52568-z.

Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing.

Rohm D, Black J, McCutcheon S, Barrera A, Morone D, Nuttle X bioRxiv. 2024; .

PMID: 38496583 PMC: 10942373. DOI: 10.1101/2024.03.03.583177.

References

Manguso R, Pope H, Zimmer M, Brown F, Yates K, Miller B . In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature. 2017; 547(7664):413-418. PMC: 5924693. DOI: 10.1038/nature23270. View

Guo H, Zhu P, Wu X, Li X, Wen L, Tang F . Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 2013; 23(12):2126-35. PMC: 3847781. DOI: 10.1101/gr.161679.113. View

Korkmaz G, Lopes R, Ugalde A, Nevedomskaya E, Han R, Myacheva K . Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat Biotechnol. 2016; 34(2):192-8. DOI: 10.1038/nbt.3450. View

Morgan S, Mariano N, Bermudez A, Arruda N, Wu F, Luo Y . Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. Nat Commun. 2017; 8:15993. PMC: 5511349. DOI: 10.1038/ncomms15993. View

Wang K, Chang H . Molecular mechanisms of long noncoding RNAs. Mol Cell. 2011; 43(6):904-14. PMC: 3199020. DOI: 10.1016/j.molcel.2011.08.018. View

Barrangou R, Doudna J . Applications of CRISPR technologies in research and beyond. Nat Biotechnol. 2016; 34(9):933-941. DOI: 10.1038/nbt.3659. View

Maeder M, Linder S, Cascio V, Fu Y, Ho Q, Joung J . CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013; 10(10):977-9. PMC: 3794058. DOI: 10.1038/nmeth.2598. View

Gao L, Cox D, Yan W, Manteiga J, Schneider M, Yamano T . Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol. 2017; 35(8):789-792. PMC: 5548640. DOI: 10.1038/nbt.3900. View

Kuscu C, Parlak M, Tufan T, Yang J, Szlachta K, Wei X . CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations. Nat Methods. 2017; 14(7):710-712. DOI: 10.1038/nmeth.4327. View

10.

Kleinstiver B, Pattanayak V, Prew M, Tsai S, Nguyen N, Zheng Z . High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016; 529(7587):490-5. PMC: 4851738. DOI: 10.1038/nature16526. View

11.

Chavez A, Scheiman J, Vora S, Pruitt B, Tuttle M, Iyer E . Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015; 12(4):326-8. PMC: 4393883. DOI: 10.1038/nmeth.3312. View

12.

Rosenbluh J, Xu H, Harrington W, Gill S, Wang X, Vazquez F . Complementary information derived from CRISPR Cas9 mediated gene deletion and suppression. Nat Commun. 2017; 8:15403. PMC: 5457492. DOI: 10.1038/ncomms15403. View

13.

Ran F, Cong L, Yan W, Scott D, Gootenberg J, Kriz A . In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-91. PMC: 4393360. DOI: 10.1038/nature14299. View

14.

Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi N . CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature. 2014; 514(7522):380-4. PMC: 4199937. DOI: 10.1038/nature13589. View

15.

Fulco C, Munschauer M, Anyoha R, Munson G, Grossman S, Perez E . Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science. 2016; 354(6313):769-773. PMC: 5438575. DOI: 10.1126/science.aag2445. View

16.

Datlinger P, Rendeiro A, Schmidl C, Krausgruber T, Traxler P, Klughammer J . Pooled CRISPR screening with single-cell transcriptome readout. Nat Methods. 2017; 14(3):297-301. PMC: 5334791. DOI: 10.1038/nmeth.4177. View

17.

Macosko E, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M . Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. 2015; 161(5):1202-1214. PMC: 4481139. DOI: 10.1016/j.cell.2015.05.002. View

18.

Zhu S, Li W, Liu J, Chen C, Liao Q, Xu P . Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library. Nat Biotechnol. 2016; 34(12):1279-1286. PMC: 5592164. DOI: 10.1038/nbt.3715. View

19.

Creyghton M, Cheng A, Welstead G, Kooistra T, Carey B, Steine E . Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc Natl Acad Sci U S A. 2010; 107(50):21931-6. PMC: 3003124. DOI: 10.1073/pnas.1016071107. View

20.

Zetsche B, Gootenberg J, Abudayyeh O, Slaymaker I, Makarova K, Essletzbichler P . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163(3):759-71. PMC: 4638220. DOI: 10.1016/j.cell.2015.09.038. View