Endogenous spacing enables co-processing of microRNAs and efficient combinatorial RNAi

Alexandra M Amen; Ryan M Loughran; Chun-Hao Huang; Rachel J Lew; Archna Ravi; Yuanzhe Guan; Emma M Schatoff; Lukas E Dow; Brooke M Emerling; Christof Fellmann

doi:10.1016/j.crmeth.2022.100239

Endogenous spacing enables co-processing of microRNAs and efficient combinatorial RNAi

Cell Rep Methods. 2022 Jun 21;2(7):100239. doi: 10.1016/j.crmeth.2022.100239. eCollection 2022 Jul 18.

Authors

Alexandra M Amen^{1

2}, Ryan M Loughran³, Chun-Hao Huang^{4

5}, Rachel J Lew¹, Archna Ravi³, Yuanzhe Guan⁶, Emma M Schatoff^{5

7}, Lukas E Dow^{7

8}, Brooke M Emerling³, Christof Fellmann^{1

6

9}

Affiliations

¹ Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
³ Cell and Molecular Biology of Cancer Program, Sanford Burnham Prebys, La Jolla, CA, USA.
⁴ Memorial Sloan Kettering Cancer Center, New York, NY, USA.
⁵ Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medicine, New York, NY, USA.
⁶ Mirimus Inc., Brooklyn, NY, USA.
⁷ Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.
⁸ Department of Biochemistry, Weill Cornell Medicine, New York, NY, USA.
⁹ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.

Abstract

We present Multi-miR, a microRNA-embedded shRNA system modeled after endogenous microRNA clusters that enables simultaneous expression of up to three or four short hairpin RNAs (shRNAs) from a single promoter without loss of activity, enabling robust combinatorial RNA interference (RNAi). We further developed complementary all-in-one vectors that are over one log-scale more sensitive to doxycycline-mediated activation in vitro than previous methods and resistant to shRNA inactivation in vivo. We demonstrate the utility of this system for intracranial expression of shRNAs in a glioblastoma model. Additionally, we leverage this platform to target the redundant RAF signaling node in a mouse model of KRAS-mutant cancer and show that robust combinatorial synthetic lethality efficiently abolishes tumor growth.

Keywords: RNA interference; RNAi; cancer modeling; combinatorial target inhibition; gene regulation; microRNA; target discovery; target validation; viral vectors.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Animals
Genetic Vectors
Mice
MicroRNAs* / genetics
Promoter Regions, Genetic
RNA Interference
RNA, Small Interfering / genetics

Substances

MicroRNAs
RNA, Small Interfering

Abstract

Publication types

MeSH terms

Substances

Grants and funding