Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: A brief overview.
Recently the Cas9, an RNA guided DNA endonuclease, emerged as a powerful tool for targeted genome manipulations. Cas9 protein can be reprogrammed to cleave, bind or nick any DNA target by simply changing crRNA sequence, however a short nucleotide sequence, termed PAM, is required to initiate crRNA hybridization to the DNA target. PAM sequence is recognized by Cas9 protein and must be determined experimentally for each Cas9 variant. Exploration of Cas9 orthologs could offer a diversity of PAM sequences and novel biochemical properties that may be beneficial for genome editing applications. Here we briefly review and compare Cas9 PAM identification assays that can be adopted for other PAM-dependent CRISPR-Cas systems.
Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.
Tautvydas
T
Karvelis
Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.
Giedrius
G
Gasiunas
Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania. Electronic address: siksnys@ibt.lt.
Virginijus
V
Siksnys
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019295:Computational Biology; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D015723:Gene Library; D015894:Genome, Human; D057166:High-Throughput Screening Assays; D006801:Humans; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
CRISPR; Cas9; Genome editing; High-throughput methods; PAM library