CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies

Rebecca Davies,Catherine Itman,Aditi Qamra,Sheng Taotao,Amit Mandoli,Richa Chaturvedi,Joseph Rosenbluh,Ling Liu,Changyuan Hu,Kie Kyon Huang,Patrick Tan,David Powell,Natasha Tuano,Roger J Daly

doi:10.1186/s13059-021-02266-6

Abstract

IntroductionGenes contain multiple promoters that can drive the expression of various transcript isoforms. Although transcript isoforms from the same gene could have diverse and non-overlapping functions, current loss-of-function methodologies are not able to differentiate between isoform-specific phenotypes.ResultsHere, we show that CRISPR interference (CRISPRi) can be adopted for targeting specific promoters within a gene, enabling isoform-specific loss-of-function genetic screens. We use this strategy to test functional dependencies of 820 transcript isoforms that are gained in gastric cancer (GC). We identify a subset of GC-gained transcript isoform dependencies, and of these, we validate CIT kinase as a novel GC dependency. We further show that some genes express isoforms with opposite functions. Specifically, we find that the tumour suppressor ZFHX3 expresses an isoform that has a paradoxical oncogenic role that correlates with poor patient outcome.ConclusionsOur work finds isoform-specific phenotypes that would not be identified using current loss-of-function approaches that are not designed to target specific transcript isoforms.

Highlights

Genes contain multiple promoters that can drive the expression of various transcript isoforms
Others and us have shown that CRISPR interference (CRISPRi)-induced phenotypes are only observed when dCas9-KRAB is targeted to a defined region that is located ± 150 bp from the transcriptional start site (TSS) [16, 18]
We found that sgRNAs targeting promoter P1 had an effect only on HNF4A isoform 1 expression (Fig. 1b), and sgRNA targeting promoter P2 inhibited only HNF4A isoform 2 expression (Fig. 1c)

Summary

Introduction

Genes contain multiple promoters that can drive the expression of various transcript isoforms. Two main mechanisms drive the generation of transcript isoforms: alternative splicing and differential promoter usage. Differential promoter usage is a mechanism by which alternative promoters within a gene drive the expression of different transcript isoforms [1]. RNA-Seq in conjugation with ChIP-Seq profiling in human cells has shown that the majority of transcript isoforms are produced by differential promoter usage rather than alternative splicing [5]. Consistent with these observations, most genes in the human genome contain multiple promoters that have the ability to transcribe various isoforms [6, 7]. In addition to regulating when a gene is transcribed, promoters regulate which isoforms are expressed

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Conclusion