Derived from adaptive immune systems of a microbiome, CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 genome editing tool cuts the target DNA region and allows the DNA to be repaired naturally. It is also known as "RGENs" (RNA-guided engineered nuclease), as it consists of guide RNA (gRNA) and Cas9 nuclease. This technology provides easier and more efficient ways for precise manipulation of genomes. gRNA consists of crRNA and tracrRNA. The crRNA has a 20-nt target-complementary sequence, while the tracrRNA has the recognition sequence necessary for Cas9-binding (Figure 1). The gRNA guides Cas9 nuclease to the target DNA region, which binds to the complementary sequence in the target DNA region. During this step, the target DNA must contain a PAM (Protospacer adjacent motif) sequence at the 3’-end. After the gRNA forms a complex with the Cas9 nuclease and binds to the target DNA region with the complemtary base pairing, the Cas9 nuclease will recognize the PAM sequence, which results in DSBs at the 3 bp upstream of the PAM sequence (Figure 2).
|Figure 1. Diagram of gRNA and Cas9 nuclease complex
The gRNA consists of crRNA and tracrRNA. While the former contains a 20-nt target-complementary sequence, the latter contains a scaffold sequence allowing to form a complex with the Cas9 nuclease.
|Figure 2. A schematic diagram of CRISPR-Cas9 System
RNA-guided Cas9 nuclease recognizes the PAM sequence (5'-'NGG'-3') at the 3'-end of the target DNA region and creates DSBs at the 3 bp upstream of the PAM sequence.
DSBs are repaired by means of two pathways: Non-homologous end-joining (NHEJ) or Homology-directed repair (HDR) pathways. If a donor template is not present, DSBs will be repaired through NHEJ pathway. The NHEJ pathway causes a gene knock-out by frameshifts and premature stop codons created from the indels, insertions and deletions of bases in a gene.
On the other hand, when donor templates are present, HDR pathway will take place. Compared with the other case, they can provide more accurate modification and insertion of new genes (Figure 3).
|Figure 3. A step-by-step diagram showing CRISPR-Cas9-mediated DSBs repair mechanisms
There are two types of pathways for repairing DSBs: NHEJ and HDR. NHEJ pathway creates an indels, variations by insertions and deletions of few bases during the repair of DSB’s terminal. On the other hand, the HDR pathway uses homologous sequences of donors, either in the form of dsDNA or ssDNA, which allows more accurate gene insertion than the other.