Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

Abstract

CRISPR-based gene activation (CRISPRa) is a promising therapeutic approach for gene therapy, upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically and potently upregulating target genes, including autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate therapeutically relevant genes in a cell type-specific manner.

Chromatin context-dependent regulation and epigenetic manipulation of prime editing

“…Although recent advances have shown that this task is tractable 60 , more work is in demand to achieve higher recovery of feature barcodes in sci-RNA-seq3 experiments, which critically underlies the scalability of this method. Finally, in the CRISPRa experiments, to achieve enduring activation of target genes, we utilized a cell line with integrated dCas9-VP64 57 , which could potentially compete with the prime editor for pegRNA binding, resulting in reduced concentrations of functional pegRNA and physical hindrance at prime editing target sites. Alternative strategies such as using in vitro assembled prime editing RNPs or orthogonal CRISPRa systems (e.g.…”

“…For gene activation, we chose the CRISPRa Synergistic Activation Mediator (SAM) system 56 . On Day 0, we transfected a K562 cell line stably expressing the dCas9-VP64 fusion 57 with a pair of gRNAs (2XMS2) targeting the promoter of one of these four genes, along with an MCP-p65-Rta fusion protein. On Day 2, we transfected the cells with PEmax and pegRNAs to introduce the desired mutations (Fig.…”

Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus

“…To reiterate, the primary aim of our platform development has been to enable the discovery of novel endogenous regulators of therapeutic relevance, and then translate these native genes to common ORFs or rationally-designed synthetic genes, intended for CAR or other forms of adoptive T cell therapy. However, given their modularity and capacity for multiplex gene modulation, 73 we envision that CRISPRa + T cells could conceivably be offered, either as stand-alone or programmed, personalized GoF treatment options in due course. Indeed, private pharmaceutical companies have been founded, predicted on the development of in vivo CRISPRa drugs.…”

Optimization of Cas12a for multiplexed genome-scale transcriptional activation

“…The technology described here should likewise easily extend to combinatorial screens, such as activating multiple genes at their respective promoters, an approach that is extraordinarily useful to engineer cell types of interest and understand cell fate decisions. Further, scalable combinatorial screens can enable dissection of the regulatory logic of the non-coding genome, by jointly targeting potential enhancer elements and putative promoter targets 16,45 . In sum, we have demonstrated methods for implementing Cas12a-based CRISPRa at scale in human cells.…”