Haemophilus parainfluenzae Cas9 PAM discovery
A dsDNA target library with randomized PAM sequences was generated by overlapping PCR, with theforward primer containing the 10-nt randomized PAM region. The library was gel-purified and subjected toin vitro cleavage reaction by purified Cas9 along with T7-transcribed sgRNAs.The reaction was then treated with proteinase K at 50C for 10 minutes and run on a 4% agarose/1xTAEgel. The cleavage product was excised, eluted, and cloned using a previously described protocol (Zhanget al., 2012), with modifications. Briefly, DNA ends were repaired, non-templated 2-deoxyadenosine tailswere added, and Y-shaped adapters were ligated. After PCR, the product was quantitated with KAPALibrary Quantification Kit and sequenced using a NextSeq 500 (Illumina) to obtain 75 nt paired-end reads.The 10-nt randomized PAM region is followed by GGATCCTCTAGAGTCGACCTGCAGG.