Genome engineering and disease modeling via programmable nucleases for insulin gene therapy: Promises of CRISPR/Cas9 technology

Figure 2 Applications of CRISPR/Cas9 technology. CRISPR/Cas9 system, as a result of rapidly developing technology, has become the most commonly used tool in almost all gene editing fields today. The Cas9 enzyme is used for many different purposes by subjecting it to various changes and fusing it with a series of effective proteins[112]. Catalytically inactive Cas9 (dCas9) can be attached to numerous transcription-modifiable enzymes to achieve epigenetic and transcriptional control of targeted genes. Transcription activators are recruited to control gene expression using the CRISPR-based activators SunTag, viral protein regulatory (VPR), and synergistic activator mediator (SAM). Conventional peptide linkers are used by VPR to attach the tripartite VP64, p65, and Rta effector to dCas9. MCPs linked to a p65-HSF1 domain are directed by the SAM using the MS2 RNA aptamer to induce transcription. In the SunTag system, a GCN4-epitope array is used to recruit VP64 activators to transcription start sites. Transcription can be inhibited by the dCas9-KRAB via deploying similar strategies. CpG dinucleotides can be de-novo methylated by the DNA methyltransferase 3A (DNMT3A) attached to dCas9 in a programmable manner. Methylated CpG sites can be demethylated via ten-eleven translocation’s (TET1) catalytic domain linked to dCas9. By aiming CpG-containing promoter regions for epigenetic alteration, dCas9-DNMT3A/TET1 can successfully control gene transcription. The human p300 acetyltransferase (p300core) I or histone deacetylase 8 linked to dCas9 can regulate the acetylation status of histone 3 Lysine 27 residues to control transcription from promoters and enhancers. Moreover, in recent years, it has been used with base regulatory proteins for base editing and fluorescent proteins such as GFP to monitor a specific region on the chromosome. In another application, the nuclear organization of chromosomes can be rearranged in 3D in a temporary and inducible manner (CRISPR GO). dCas9: dead Cas9; SAM: Synergistic activator mediator; DNMT: DNA methyltransferase; ABE: Adenine base editor; CBE: Cytosine base editor; GFP: Green fluorescent protein; VPR: Viral protein regulatory.