Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

Yang Zhang; Tuan M Nguyen; Xiao-Ou Zhang; Limei Wang; Tin Phan; John G Clohessy; Pier Paolo Pandolfi

doi:10.1186/s13059-021-02263-9

Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

Genome Biol. 2021 Jan 21;22(1):41. doi: 10.1186/s13059-021-02263-9.

Authors

Yang Zhang^{1

2

3}, Tuan M Nguyen^#^{1

2

4}, Xiao-Ou Zhang^#⁵, Limei Wang^{1

2}, Tin Phan^{1

2}, John G Clohessy^{6

7

8}, Pier Paolo Pandolfi^{9

10

11

12}

Affiliations

¹ Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215, USA.
² Ludwig Center at Harvard, Harvard Medical School, Boston, MA, 02215, USA.
³ Present address: Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA, 02215, USA.
⁴ Present address: Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.
⁵ Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
⁶ Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215, USA. jclohess@bidmc.harvard.edu.
⁷ Ludwig Center at Harvard, Harvard Medical School, Boston, MA, 02215, USA. jclohess@bidmc.harvard.edu.
⁸ Preclinical Murine Pharmacogenetics Facility and Mouse Hospital, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215, USA. jclohess@bidmc.harvard.edu.
⁹ Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215, USA. PierPaolo.PandolfiDeRinaldis@renown.org.
¹⁰ Ludwig Center at Harvard, Harvard Medical School, Boston, MA, 02215, USA. PierPaolo.PandolfiDeRinaldis@renown.org.
¹¹ Department of Molecular Biotechnology and Health Sciences, University of Turin, 10126, Turin, Italy. PierPaolo.PandolfiDeRinaldis@renown.org.
¹² Renown Institute for Cancer, Nevada System of Higher Education, Reno, NV, 89502, USA. PierPaolo.PandolfiDeRinaldis@renown.org.

^# Contributed equally.

Abstract

Short hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.

Keywords: CRISPR/Cas13d system; High-throughput screening; circRNAs.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats*
High-Throughput Screening Assays
Humans
RNA / genetics*
RNA Splicing
RNA, Circular / genetics*
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Small Interfering

Substances

RNA, Circular
RNA, Guide, CRISPR-Cas Systems
RNA, Small Interfering
RNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding