CasZyme team were able to characterize 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components revealed additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target.
Human therapeutics, Diagnostics, Industrial Biotechnologies.
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