Genome-Scale Perturbation of Long Noncoding RNA Expression Using CRISPR Interference

S John Liu; Max A Horlbeck; Jonathan S Weissman; Daniel A Lim

doi:10.1007/978-1-0716-1158-6_20

Genome-Scale Perturbation of Long Noncoding RNA Expression Using CRISPR Interference

Methods Mol Biol. 2021:2254:323-338. doi: 10.1007/978-1-0716-1158-6_20.

Authors

S John Liu^{1

2}, Max A Horlbeck^{3

4

5

6}, Jonathan S Weissman^{3

4

5

6}, Daniel A Lim^{7

8

9}

Affiliations

¹ Department of Neurological Surgery, University of California, San Francisco, CA, USA.
² Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA, USA.
³ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA.
⁴ Howard Hughes Medical Institute, University of California, San Francisco, CA, USA.
⁵ California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA, USA.
⁶ Center for RNA Systems Biology, University of California, San Francisco, CA, USA.
⁷ Department of Neurological Surgery, University of California, San Francisco, CA, USA. Daniel.Lim@ucsf.edu.
⁸ Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA, USA. Daniel.Lim@ucsf.edu.
⁹ San Francisco Veterans Affairs Medical Center, University of California, San Francisco, CA, USA. Daniel.Lim@ucsf.edu.

Abstract

CRISPR-mediated interference (CRISPRi), a robust and specific system for programmably repressing transcription, provides a versatile tool for systematically characterizing the function of long noncoding RNAs (lncRNAs). When used with highly parallel, lentiviral pooled screening approaches, CRISPRi enables the targeted knockdown of tens of thousands of lncRNA-expressing loci in a single screen. Here we describe the use of CRISPRi to target lncRNA loci in a pooled screen, using cell growth and proliferation as an example of a phenotypic readout. Considerations for custom lncRNA-targeting libraries, alternative phenotypic readouts, and orthogonal validation approaches are also discussed.

Keywords: CRISPR; CRISPRi; Screen; lncRNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems
Cell Line, Tumor
Cell Proliferation
Gene Knockdown Techniques / methods*
HEK293 Cells
Humans
Lentivirus / genetics
Lentivirus / physiology*
Promoter Regions, Genetic
RNA, Long Noncoding / genetics*
Transcription, Genetic

Substances

RNA, Long Noncoding

Abstract

Publication types

MeSH terms

Substances

Grants and funding