Extended Data Fig. 1: Targeting range and characterization of previous engineered SpCas9 PAM variant enzymes.
From: Custom CRISPR–Cas9 PAM variants via scalable engineering and machine learning
(a) Quantification of pathogenic and likely pathogenic single nucleotide variants (SNVs) from ClinVar98 that are theoretically revertible using ABE or CBE based on their proximity to an NGG PAM. SNVs were considered editable if a GG dinucleotide PAM was available at the appropriate distance upstream on the correct DNA strand, positioning the SNV anywhere between positions 5–9 of the spacer sequence (counting from the PAM-distal end of the spacer; typically called the ‘edit window’ of base editors). (b-d) Heatmap representations of the PAM profiles of SpCas9 enzymes determined using the HT-PAMDA assay3,46, for wild-type SpCas9 (panel b), for enzymes with altered PAM requirements (e.g. SpCas9-VRQR14,42, SpCas9-VRER14, and xCas9 (ref. 2); panel c), and for enzymes with relaxed PAM requirements (e.g. SpCas9-NG1, SpG and SpRY3, and SpCas9-NRRH/NRCH/NRTH4); panel d). The log10 rate constants (k) are the mean of n = 2 replicate HT-PAMDA experiments performed using two distinct spacer sequences. Because the HT-PAMDA assay measures the relative depletion of substrates encoding various PAMs, it may underestimate rate constants for enzymes with highly relaxed PAM requirements such as SpRY and Cas9-NRRH.
