CRISPR Interact

Adaptation

The CRISPR system is designed to provide immunity for bacteria after the invasion of a bacteriophage (hereafter: foreign element). The first step of all CRISPR systems, including Type II CRISPR systems, is the Adaptation phase (Richter et al. 2012). The adaptation phase is very complex and although much has been studied of the mechanisms behind the phase, it is the least studied of all three phases (Loureriro et al. 2019).

 

 

There are two essential proteins for the adaptation phaseーCas1 and Cas2. SpyCas1 is displayed in the Jsmol interface. These genes for these proteins are located upstream of the CRISPR array. These proteins are necessary for a process called “spacer acquisition,” where the proteins acquire specific parts of the foreign element, called protospacers, that are inserted into the CRISPR array as spacers. 

To achieve this, Cas1 and Cas2 both have nuclease activity. Mutations in Cas1 and Cas2 can stop the CRISPR system as a whole (Loureriro et al. 2019). We can artificially create mutations win the amino acid in Jsmol. 

Start by opening the console in Jsmol and clicking on a random amino acid in the interface. Take note of the amino acid’s three-letter codon (see chart below) and its residue number as shown below. Now type mutate [residue number] [desired amino acid] to change that amino acid to the desired amino acid. Now type select [residue number] [original amino acid] to check if the mutation has been completed. If the terminal returns 0 atoms selected, then it worked. Note that this does not always equate to a complete loss of function of the CRISPR system. Sometimes, if the amino acid has similar properties to the previous protein, then there may not be a loss of function.

 

 

Both Cas1 and Cas2 are homodimers, which mean they consist of one protein. Two of each protein come together to form a heterotetramer. You can view this protein in a Type II-A CRISPR system by typing 6QXF. You can select the domains of each of the two individual Cas1/Cas2 proteins by typing select chain :[chain Letter]. The chain’s letter should be shown when you first opened the PDB of the complex. Try to use this website to identify each Cas1/Cas2 website using restrict :[chain letter 1] :[chain letter 2]. Also try to identify the DNA in the complex.

 

To continue, the specific parts of the foreign element mentioned earlier are not chosen randomly. Rather, the complex recognizes and selects a certain part of the foreign element, called the protospacer adjacent motif (PAM), made from a short sequence of DNA bases, usually 2-6 bp (Loureriro et al. 2019). The PAM sequence for the S. pyogenes CRISPR system is 5'-NGG-3', where N is any base and G is guanine. Then, the complex will make cuts in the foreign element to create a protospacer (20-50 bp (Brooker 2018)) then finally guide and incorporate the protospacer into the CRISPR array as a spacer. By cutting the foreign element, the CRISPR system also inactivates the DNA (Brooker 2018).

 

Even after heavy research into the adaptation phase, much of the processes, especially protospacer incorporation, behind it has been relatively unknown.