Abstract
Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq) which leverages the RNA-targeting CRISPR/Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable array which is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.
Competing Interest Statement
In the past three years, RS has worked as a consultant for Bristol-Myers Squibb, Regeneron, and Kallyope, and served as a SAB member for ImmunAI, Apollo Life Sciences GmbH, Nanostring, and the NYC Pandemic Response Lab. NES is an advisor to Vertex. PS is a co-inventor on a patent related to protein detection by sequencing as described in this work. The New York Genome Center and New York University have applied for patents relating to the work in this article.
