Cell Reprogramming With CRISPR/Cas9 Based Transcriptional Regulation Systems

Abstract

The speed of reprogramming technologies evolution is rising dramatically in modern science. Both the scientific community and health workers depend on such developments due to the lack of safe autogenic cells and tissues for regenerative medicine, genome editing tools and reliable screening techniques. To perform experiments efficiently and to propel the fundamental science it is important to keep up with novel modifications and techniques that are being discovered almost weekly. One of them is CRISPR/Cas9 based genome and transcriptome editing. The aim of this article is to summarize currently existing CRISPR/Cas9 applications for cell reprogramming, mainly, to compare them with other non-CRISPR approaches and to highlight future perspectives and opportunities.

Keywords: CRISPR/Cas9; dCas9; genome screening; human cell reprogramming; regenerative medicine; transactivator systems.

FIGURE 1

CRISPR/Cas9-based systems. (A) Classical CRISPR/Cas9 system, where sgRNA guided Cas9 performs a double-strand break in target locus. In order to manipulate gene expression the dCas9 mutant without nucleolytic activity was created. First generation CRISPR/dCas9 systems consist of two components: dCas9 fused with effector and sgRNA. They include (B) chimeric dCas9, which can physically block RNA polymerase and repress elongation or, if fused with effectors, activate or repress gene expression; and (C) VPR, where three activator domains VP64, p65, Rta are fused with dCas9.

FIGURE 2

Second generation CRISPR/dCas9 systems, which activation or repression is amplified due to recruiting multiple copies of an effector by RNA. (A) Scaffold and (B) Casilio systems use RNA regions [aptamers in Scaffold and PUF-binding site (PBS) in Casilio] for simultaneous recruitment of different effectors to different sgRNAs by RNA binding proteins (RPB in Scaffold and PUF in Casilio). (C) in SAM RNA binding protein MS2 is fused with two activator domains, p65 and HSF1, and dCas9 is also fused with activator VP64.

FIGURE 3

Second generation CRISPR/dCas9 system SunTag, in which multiple copies of GCN4 recruit multiple molecules of the chimeric scFv antibody-activator protein to the target site.

FIGURE 4

Second generation CRISPR/dCas9 system TREE, which is a combination of Scaffold and SunTag, where (GCN4)n tail binds with RNA aptamer by MS2 RNA binding protein.