Dual gRNA screen
Aim
Cell states are governed by complex heterotypic interactions between regulatory genes. Previous work has used genetic loss of function to study the complex interdependent roles of transcription factors controlling gene expression, differentiation, and morphogenesis. We have developed an industry partnership with Agilent to establish a scalable screening platform using standard CRISPR editing strategies.
Brief project outline
We will screen the transcriptional regulatory dependencies that underlie all possible pairwise perturbations of up to 20 different heterotypic regulatory genes in the first instance. We will leverage single cell bar-coding systems to perform scalable sequencing of samples using platforms developed at the WEHI (in collaboration with Shalin Naik).
Genomics-based innovative aspect of proposal
While previous studies have been limited to analysis of 1-3 regulatory genes in a combinatorial manner, in this project we aim to utilize scalable bar-coded RNA-seq with a library of multiplexed dual-gRNA gene knockouts to systematically map the heterotypic roles and interdependencies of transcription factors and epigenetic regulators controlling cell identity.
Broad applicability of the technique
The outcome of this project will enable two new platforms at UQ:
- A platform for arrayed CRISPR screening using multiple synthetic guide RNA.
- A platform for Cell-seq2 based bulk RNA-seq.
Publications arising from this project
Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle (https://doi.org/10.1083/jcb.201905065)
Therapeutic Inhibition of Acid Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury (https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.121.054360)
