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

被引：111

作者：

Zhang, Yang ^{[1
,2
,3
]}

Nguyen, Tuan M. ^{[1
,2
,4
]}

Zhang, Xiao-Ou ^{[5
]}

Wang, Limei ^{[1
,2
]}

Phan, Tin ^{[1
,2
]}

Clohessy, John G. ^{[1
,2
,6
,7
]}

Pandolfi, Pier Paolo ^{[1
,2
,8
,9
]}

机构：

[1] Harvard Med Sch, Beth Israel Deaconess Canc Ctr, Dept Med & Pathol, Beth Israel Deaconess Med Ctr,Canc Res Inst, Boston, MA 02215 USA

[2] Harvard Med Sch, Ludwig Ctr Harvard, Boston, MA 02215 USA

[3] Harvard Med Sch, Sect Integrat Physiol & Metab, Joslin Diabet Ctr, Boston, MA 02215 USA

[4] Broad Inst MIT & Harvard, Chem Biol & Therapeut Sci, Cambridge, MA 02142 USA

[5] Univ Massachusetts, Program Bioinformat & Integrat Biol, Sch Med, Worcester, MA 01605 USA

[6] Harvard Med Sch, Preclin Murine Pharmacogenet Facil, Beth Israel Deaconess Med Ctr, Boston, MA 02215 USA

[7] Harvard Med Sch, Mouse Hosp, Beth Israel Deaconess Med Ctr, Boston, MA 02215 USA

[8] Univ Turin, Dept Mol Biotechnol & Hlth Sci, I-10126 Turin, Italy

[9] Nevada Syst Higher Educ, Renown Inst Canc, Reno, NV 89502 USA

来源：

GENOME BIOLOGY | 2021年 / 22卷 / 01期

关键词：

circRNAs; CRISPR; Cas13d system; High-throughput screening; PROFILING REVEALS; ESSENTIAL GENES; CIRCULAR RNAS; IDENTIFICATION; SORAFENIB; ABUNDANT; CRISPR; GUIDE;

D O I：

10.1186/s13059-021-02263-9

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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.

引用

页数：22

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