CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated) is an RNA-guided endonuclease that uses RNA-DNA complementarity to precisely target and cleave double-stranded DNA. To find its target, Cas9 must meticulously search double-stranded DNA across the entire genome to identify regions of gRNA (guide RNA) complementarity. This search is simplified via the protospacer-adjacent motif (PAM) located immediately next to the targeted sequence. The PAM acts as a rapid and efficient initial filter for possible target sites, circumventing the slow, kinetically unfavorable process of initiating duplex melting and R-loop formation. In the case of Streptococcus pyogenes Cas9 (henceforth referred to as Cas9), the required PAM sequence is NGG (where N can represent any nucleotide and G signifies guanine). In its native context, the PAM also serves as a mechanism to distinguish between self and non-self sequences. For genome editing applications, the PAM requirement imposes a major limitation on the number of sequences that can be targeted. Protein engineering efforts have focused on re-targeting or reducing the PAM specificity. A PAMless Cas9 variant, termed SpRY, is different in that it is capable of targeting nearly any site in vivo. SpRY binds diverse PAM sequences by forming non-specific interactions with the phosphodiester backbone of the target DNA. The non-specific interactions employed by SpRY causes the enzyme to accumulate at off-target sites where DNA melting has initiated, but the R-loop cannot complete due to the lack of complementarity. Here you have the cryoEM structure of SpRY Cas9 in complex with the gRNA and a DNA target (PDB code: 8U3Y)

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Structure rendered with @proteinimaging, post-processed with @stylar.ai_official and depicted with @corelphotopaint