Fig. 4.

Major strategies to recruit DNA- and chromatin-targeting and modifying enzymes via the CRISPR-Cas system. The schematics show various strategies of recruiting effector proteins to a target site using RNA guidable DNA binding capacity of Cas9-sgRNA complex. Effector proteins can be directly fused to active Cas9 or catalytically inactive dCas9 through a linker peptide. Additionally, the sgRNA scaffold can be engineered to contain multiple RNA aptamers that specifically bind to a known RNA binding proteins (RBP) such as MCP or PCP. Effector proteins than can be guided to a target locus by fusing them to the RBPs. In the Tripartite strategy, multiple different effectors are being recruited through dCas9 as well as engineered sgRNA scaffold. The SunTag approach utilizes a repeating peptide array of protein scaffold to recruit multiple copies of an antibody-fused effector protein. Chemically inducible strategies enable temporal control over the activity of Cas9 or Cas9 fused effector proteins. In split Cas9, each halves of Cas9 protein can be induced to form functional complex. In the intein-Cas9 approach, the intein protein segment can be chemically induced to excise from Cas9 and result in its activation