Question

What is dCas9? What is its role?

Answer 1

Ans:

DCas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease associated with the CRISPR (clustered regularly interspaced short palindromic repeats) type II adaptive immunity system in Streptococcus pyogenes, among other bacteria.

Role: The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. The Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double strand breaks in DNA. These breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity.

Due to the unique ability of Cas9 to bind to essentially any complement sequence in any genome, researchers wanted to use this enzyme to repress transcription of various genomic loci. To accomplish this, the two crucial catalytic residues of the RuvC and HNH domain can be mutated to alanine abolishing all endonuclease activity of Cas9. The resulting protein coined ‘dead’ Cas9 or ‘dCas9’ for short, can still tightly bind to dsDNA. This catalytically inactive Cas9 variant has been used for both mechanistic studies into Cas9 DNA interrogative binding and as a general programmable DNA binding RNA-Protein complex.

Principle: CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. Recently, the type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been engineered to function in eukaryotic systems using two molecular components: a single Cas9 protein and a non-coding guide RNA (gRNA). The Cas9 endonuclease can be programmed with a gRNA, directing a DNA double-strand break (DSB) at a desired genomic location. Similar to DSBs induced by zinc finger nucleases (ZFNs), the cell then activates endogenous DNA repair processes, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), to heal the targeted DSB.

Recombinant Cas9 proteins (∼160 KD) are ready-to-use reagents for genome engineering experiments. When combined with target-specific guide RNAs, Cas9 proteins will form ready-to-use nucleases suitable for transfection of cell cultures and for the accelerated development of genetically-modified animals via one-cell embryo injection.