There are three ways of introducingCRISPR-Cas componentsinto the cells:

a) Using aCRISPR DNA vector: this is the most common way as it is cheap and relatively efficient for any experiment. Both gRNA and Cas9 DNA sequences are part of a plasmid, which can be introduced into the cells via transfection or transduction. Once inside the nucleus, it follows the standard process of transcription and translation until the gRNA and the Cas protein form the RNP complex. Then, thanks to a Nucleus Localization Signal, also included in the plasmid, the RNP will go inside the nucleus to bind the target DNA sequence.

b) Using amRNA/gRNA mix: both RNAs sequences are co-transfected into the cell cytoplasm, where mRNA is translated into the Cas protein, which then binds the gRNA to form the RNP to enter the nucleus and look for the target DNA sequence.

c)使用reagent-protein complex: the RNP is directly introduced to the cell cytoplasm using a specific reagent.