- The Question
- The Answer
- CRISPR Background
- Genome Editing
- Cas9 Mechanism
- Molecular Techniques
DNA Binding and Cleavage
CRISPR/Cas9 systems use a guide RNA with a region complementary to the target DNA to specifically bind their target sequences. However, there is an immediate and inherent issue with this. In order to achieve specificity, longer guide RNAs are beneficial, as each nucleotide in the RNA guide increases the specificity of the nuclease about 4-fold. However, in order for the DNA to melt and accommodate base-pairing to the guide RNA, the longer the RNA guide, the less efficient the nuclease. How can CRISPR/Cas9 systems have such dramatically increased specificity over other nucleases such as TALENS and ZFNS and still maintain roughly the same, if not better, efficiency? (Mali et al. 2013)
The answer is that the CRISPR/Cas9 system uses the Protospacer Adjacent Motif (PAM) binding as a preliminary step in locating the target sequence. As was determined by single molecule fluorescence microscopy, the initial binding of Cas9 to PAM (N-G-G) sequences allows the enzyme to quickly screen for potential target sequences. The enzyme will rapidly detach from DNA that does not have the proper PAM sequence. If the protein finds a potential target with the appropriate PAM, it will to melt the remaining DNA on the target to test whether the remaining target sequence is complementary to its guide sequence. The PAM binding step allows the protein to quickly screen potential targets and avoid melting many non-target sequences in its search for fully complementary sequences to cut. (Sternberg et al. 2014)
In July of 2014, Anders et al. published a crystal structure that led to a model for PAM-dependent target DNA binding, unwinding, and recognition by the Cas9 nuclease. The following images are created based off of figure 4 of the paper, or are images rendered in Pymol (distributed by Schrödinger) using the crystal structure from that paper (obtained from the Protein Data Bank).
Proposed model for PAM-dependent target DNA binding, melting, and recognition by Cas9:
1. PAM Binding:
The Protospacer Adjacent Motif (PAM) NGG bases of the target DNA strand are shown in yellow. Arginine residues 1333 and 1335 of the PAM Interacting (PI) domain bind to the major groove of the guanine bases in the PAM. A lysine residue in the Phosphate Lock Loop, also in the PI domain, binds the minor groove.
2. Phosphate Lock Loop:
This positions the PAM and target DNA such that serine 1109 in the phosphate lock loop, and two nitrogens of the phosphate lock loop’s backbone, can form hydrogen bonds to the phosphate at position +1 of the PAM. This stabilizes the target DNA such that the first bases of the target sequence (or the protospacer) can melt and rotate upwards towards the guide RNA.
3. Guide RNA:
If the target DNA is complementary to the guide RNA strand, the two strands will base pair. This will allow the target DNA to unzip, as the bases flip up and bind the guide RNA. Without the initial PAM binding and stabilization of the +1 phosphate, the guide RNA would very rarely be able to bind the target DNA, and Cas9 would be very inefficient. This illustrates a mechanism that explains why Cas9 is able to have both high efficiency and high specificity, thus making it a powerful genome editing tool.
Finally, complete annealing of the guide RNA to the target DNA allows the HNH and RuvC nucleases to cleave their respective strands. These nucleases cleave very specifically between the 3rd and 4th nucleotides from the PAM. Again, this specificity of cleavage, as well as the fact that the individual nucleases may be mutated independently and without affecting the ability of Cas9 to bind specific sequences, make the CRISPR/Cas9 system a simultaneously powerful and flexible genome editing tool.