dCas9 techniques for transcriptional repression in mammalian cells: Progress, applications and challenges

Abstract

Innovative loss-of-function techniques developed in recent years have made it much easier to target specific genomic loci at transcriptional levels. CRISPR interference (CRISPRi) has been proven to be the most effective and specific tool to knock down any gene of interest in mammalian cells. The catalytically deactivated Cas9 (dCas9) can be fused with transcription repressors to downregulate gene expression specified by sgRNA complementary to target genomic sequence. Although CRISPRi has huge potential for gene knockdown, there is still a lack of systematic guidelines for efficient and widespread use. Here we describe the working mechanism and development of CRISPRi, designing principles of sgRNA, delivery methods and applications in mammalian cells in detail. Finally, we propose possible solutions and future directions with regard to current challenges.

Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

“…CRISPRi knockdown can be tunable and reversible, making it more appropriate for some particular applications than CRISPR-del [19]. Nevertheless, the larger size of Cas9 protein fused to a repressing domain can impair its delivery to target cells and make the system less efficient [20]. Besides, CRISPRi is not suitable for targeting bidirectional promoters, as the repressive domain would induce the unspecific knockdown of all proximal TSSs [21,22], while CRISPR-del can be directed to target promoter regions with adjacent TSS with high specificity [23].…”