Structure of Cas9
Cas9 is the functional unit of CRISPR Cas9 system that locates the target gene by sliding over the DNA and when it finds the protospacer adjacent motif (PAM) sequence, it performs its catalytic activity and produces nicks or DSB in the DNA leading to the alteration in the gene sequence. Mostly these cuts are repaired by the natural mechanism of cell repair system either NHEJ or HDR. NHEJ repair system, being error-prone, produces indels causing the frameshift mutation while HDR repair pathway leads to the alteration of the gene. The function of Cas9 in bacteria is to find the foreign DNA by hovering over the external and internal genome, confirm it to be non-self DNA by recognizing PAM sequence and to cut the DNA if this contains a PAM sequence. So, the PAM sequence is of the great importance in distinguishing between the self and non-self DNA strands. The major difference in protospacer (present on viral DNA) and spacer (previous viral origin DNA incorporated into host genome) is the presence of PAM sequence that is nothing but –NGG (Yamano et al., 2017). This specific sequence is mainly present in the viral genome and not in the incorporated spacers presented between short palindromic sequences. The importance of Cas9 protein is not because of its native functionality but because of its ability to edit any genes after small genetic manipulation.
Cas9 is bi-lobed endonuclease enzymes that contain six functional domains. These domains along with their functions are REC l, REC ll, PAM interacting (PI) domain, HNH domain and RuvC domain and Bridge Helix domain. REC l domains are the largest of all domains and its function is to bind with gRNA. (Fig. 6) The actual function and role of REC ll are still under heavy research. PAM interacting domain bestows Cas9 specificity for PAM sequence thereby helping to recognize the self DNA and responsible for initiating Cas9 and target DNA binding. Bridge helix is arginine-rich region and is accountable for the initiating the cleavage process after successfully binding to the target DNA. The actual nuclease activity is conferred by two domain NHN and RuvC domains that cut any DNA complimentary to gRNA and having a PAM sequence (Fig. 6) (Khan et al., 2018).
Mechanism of CRISPR/Cas9 System
CRISPR/Cas9 gene editing is carried out using the Type ll CRISPR system. The main components of the CRISPR Cas9 is shown in table 3.
Table 3: Components of CRISPR/Cas9 gene editing tool and their corresponding activity during gene editing process.
|crRNA||Guides the Cas9 protein to the external viral DNA|
|tracrRNA||Makes a complex with crRNA forming an active complex.|
|sgRNA||a tracrRNA and at least one crRNA together known as sgRNA|
|Cas9||An endonuclease that recognizes the DNA with the help of gRNA and cleaves it (can produces single strand nicks or Double Strand Breaks- DSB). A dead or catalytically deactivated Cas9 (dCas9) can bind to DNA leading to the activation or deactivation of the genes physically hindering with the promotor region of the target gene.|
|Repair template||DNA that guides the repair process occurring in the cells and letting the insertion of a specific DNA. A small DNA which is an additional component of the CRISPR/Cas9 system that is necessary to produces insertion of specific DNA sequence. It guides the natural cellular DNA repair system to allow it to produces insertion of specific desired DNA sequence.|
CRISPR/Cas9 genes are often delivered into the target cell via plasmid vector or by transfection the cells with naked DNA. Major components are shown in the image below and also listed in the table above. The crRNAs are manufactured according to target gene and different crRNA are required for different Cas9 proteins. The crRNA have to bind only to the position where cleavage is required. Multiple crRNAs and tracrRNA are genetically engineered to produces a single gRNA.
Cas9 bi-lobed structure remain in active until the gRNA binds with it and makes it in the activated from that is called pre-target state of Cas9. This state hunts for the presence of PAM sequence in whole cell and try to locate the foreign DNA containing a complimentary sequence along with PAM. On the recognition of non-self DNA because of the presence of PAM and complimentary gRNA sequence, Cas9 active complex cleaves the breaks in the target DNA. (Fig. 7) shows different steps of CRISPR/Cas9 complex performing its gene editing activity.