Fig. 9

The functional domains of different CRISPR effectors and their applications in genome-scale screens. Multiple CRISPR effectors are accessible for disrupting coding and noncoding DNA and RNA segments. One commonly employed CRISPR effector is the CRISPR-associated 9 (Cas9) nuclease, which precisely cuts DNA at a specified target site guided by a guide RNA (gRNA). Noncoding regions can be suppressed with CRISPR interference (CRISPRi) by directing the catalytically inactive Cas9 (dCas9) to promoters and enhancer regions. It can be fused with repressor domains like methyl-CpG-binding protein 2 (MeCP2) and Krüppel-associated box (KRAB). Conversely, gene expression can be enhanced by directing dCas9 fusion proteins to regions around transcription start sites (TSSs). One approach is to fuse dCas9 with transcriptional activators such as VP64, p65, and Rta (VPR). Another method is fusing dCas9 with VP64 and using a modified single gRNA (sgRNA) to recruit the activator fusion complex MS2–p65–HSF1, collectively known as the synergistic activation modulator (SAM). In addition to targeting DNA, the Cas13 nuclease can be employed to cleave RNA at a specific site indicated by a gRNA. Furthermore, dCas9 can be combined with methyltransferases (e.g., DNA methyltransferase 3A or DNMT3A) to enable targeted DNA methylation or with proteins involved in DNA demethylation (e.g., tet methylcytosine dioxygenase 1 or TET1) to facilitate targeted DNA demethylation. Moreover, linking dCas9 to acetyltransferases like p300 or histone deacetylase proteins like histone deacetylase 3 (HDAC3) enables targeted histone acetylation or deacetylation, respectively. The design of gRNAs depends on the specific CRISPR effector and the intended targets of the CRISPR screen. When focusing on protein-coding genes, gRNAs can be designed to target either exons (using CRISPR nucleases) or regions near the transcription start site (TSS) of the gene (for CRISPRi or CRISPR activation (CRISPRa)). For saturation mutagenesis using nucleases, gRNAs are designed to target multiple noncoding regions around a gene of interest. In noncoding genome-wide screens using CRISPR nucleases, CRISPRi, or CRISPRa, gRNAs are tailored to specific genomic features like cis-regulatory elements. When silencing or amplifying noncoding RNAs with CRISPRi and CRISPRa, respectively, sgRNAs are directed to regions flanking the transcription start site (TSS) of a noncoding RNA gene. Reprinted from [11] with permission from Springer Nature