CRISPR-Cas technique components

CRISPR-Cas systems are composed of two main components:

• Single Guide RNA: Also known as gRNA or sgRNA, this synthetic RNA sequence has two parts:

  • Scaffold sequence: standard sequence needed to bind the selected Cas Kas protein.
  • Spacer sequence: a sequence of ~20 nucleotides defined by the user to target the specific DNA region to modify.

• Cas protein: Kas protein: CRISPR associated protein is an enzyme that binds the gRNA and recognizes the PAM sequence. It also modifies the DNA according to the Cas protein function. The selection of the Cas protein depends on the goal of the experiment. The common Cas protein to generate knock-out cells is Cas9, which has endonuclease activity. However, there are other types of Cas proteins that can be used in the technique, such as Cpf1 or C2c2, or even modifications of the existing ones, like dCas9 or dC2c2.

Once introduced in the cells, both components are bound in the cell cytoplasm, forming a complex known as the ribonucleoprotein complex, which is transferred into the cell nucleus via a protein called Importin.