Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi

Abstract

Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance due to lack of effective and non-toxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation, and show transient, programmable gene regulation in primary cells, including human CD34+ hematopoietic stem and progenitor cells (HSPCs) and human CD3+ T cells. We demonstrate multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.

CD5 expression by dendritic cells directs T cell immunity and sustains immunotherapy responses

“…To assess the contribution of CD5 expressed on DCs to T cell priming, we used a CRISPR gene activation approach (CRISPRa) to up-regulate CD5 expression on CD5 – DCs ( 43 ). CD5 – DCs were nucleofected with single guide RNAs (sgRNAs) that target the CD5 promoter and dCas9-VP64-p65-Rta (VPR) mRNA or dCas9-VPR mRNA alone.…”

Regulation of the CRISPR-Cas12a system by methylation and demethylation of guide RNA

“…47 In CRISPR activation technology, a non-cleavage Cas nuclease (dCas) is coupled with a transcriptional effector to regulate target gene expression. 48,49 Therefore, we further designed a demethylase-inducible CRISPR activation strategy (DICa), in which a transcriptional circuit with two plasmids was designed to demonstrate our strategy (Fig. 9A).…”

Genome editing for improving nutritional quality, post-harvest shelf life and stress tolerance of fruits, vegetables, and ornamentals

“…For this nature of dCas9, they are fused with other specific enzymes that can modify or bring the required changes in the genome. The two systems, CRISPRa/CRISPRi, also utilize nuclease-deactivated Cas9, whose catalytic domains are made disabled, and then fused to transcriptional modulators (Jensen et al, 2021).…”