  CRISPR-Cas is an adaptive immune mechanism that has been harnessed for a
variety of genetic engineering applications: the Cas9 protein recognises a
2-5nt DNA motif, known as the PAM, and a programmable crRNA binds a target DNA
sequence that is then cleaved. While off-target activity is undesirable, it
occurs because cross-reactivity was beneficial in the immune system on which
the machinery is based. Here, a stochastic model of the target recognition
reaction was derived to study the specificity of the innate immune mechanism in
bacteria. CRISPR systems with Cas9 proteins that recognised PAMs of varying
lengths were tested on self and phage DNA. The model showed that the energy
associated with PAM binding impacted mismatch tolerance, cleavage probability,
and cleavage time. Small PAMs allowed the CRISPR to balance catching mutant
phages, avoiding self-targeting, and quickly dissociating from critically
non-matching sequences. Additionally, the results revealed a lower tolerance to
mismatches in the PAM and a PAM-proximal region known as the seed, as seen in
experiments. This work illustrates the role that the Cas9 protein has in
dictating the specificity of DNA cleavage that can aid in preventing off-target
activity in biotechnology applications.
