CRISPR PAM Sequence Table
A PAM sequence is the short DNA motif a CRISPR nuclease must see next to a target before it can cut. This table lists common nucleases, their PAMs, guide spacer lengths, PAM orientation and typical cut-position rules for guide RNA design.
| Nuclease | PAM | Orientation | Spacer | Cut rule | Notes |
|---|---|---|---|---|---|
SpCas9 Streptococcus pyogenes | NGG | 3' of spacer | 20 nt | ~3 bp upstream of PAM | Most common Cas9 system; broad tool and protocol support. |
SpCas9-NG Engineered SpCas9 variant | NG | 3' of spacer | 20 nt | ~3 bp upstream of PAM | Relaxed PAM requirement; useful when NGG sites are scarce. |
SaCas9 Staphylococcus aureus | NNGRRT | 3' of spacer | 21 nt | ~3 bp upstream of PAM | Smaller Cas9 ortholog, often useful for AAV delivery. |
Cas12a / Cpf1 AsCas12a or LbCas12a | TTTV | 5' of spacer | 23-24 nt | Staggered cut distal to PAM | T-rich PAM; creates sticky ends instead of blunt Cas9 cuts. |
Reading PAM ambiguity codes
PAMs use the same IUPAC nucleotide ambiguity codes as sequence motifs. For example, NGG means any base followed by two G bases, and TTTV means TTT followed by A, C or G.
| Code | Meaning |
|---|---|
| N | A, C, G or T (any base) |
| R | A or G (purine) |
| V | A, C or G (not T) |
Frequently asked questions
What is a PAM sequence?
A PAM (protospacer-adjacent motif) is a short DNA sequence next to the target site that a CRISPR nuclease must recognize before it can bind and cut. Without the right PAM, the guide RNA will not target that site efficiently.
What PAM does SpCas9 use?
The standard Streptococcus pyogenes Cas9 (SpCas9) recognizes an NGG PAM on the genomic DNA, where N can be any base. The 20 nt guide spacer sits immediately upstream of the PAM.
Is the PAM part of the guide RNA?
No. The guide RNA spacer matches the protospacer sequence next to the PAM, but the PAM itself is present in the target DNA and is not included in the guide RNA.
How is Cas12a different from Cas9?
Cas12a recognizes a T-rich PAM on the 5' side of the spacer and makes staggered cuts. Cas9 PAMs are usually on the 3' side of the spacer and SpCas9 makes a blunt cut near the PAM.
Sources
- 1A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunityJinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E · Science 337(6096):816-821; doi:10.1126/science.1225829; PMID 22745249 · 2012The whole SpCas9 row. Verified verbatim in the full text: PAM "conforms to an NGG consensus sequence… one base pair downstream of the crRNA binding sequence" (backs PAM = NGG and orientation = 3' of spacer); "Plasmid DNA cleavage produced blunt ends at a position three base pairs upstream of the PAM sequence" (backs cut rule = ~3 bp upstream of PAM, and the FAQ's "SpCas9 makes a blunt cut near the PAM"); "the 5'-terminal 20 nucleotides of the crRNA… are available for target DNA binding" (backs spacer = 20 nt). Also backs the identical cut-rule and 20 nt spacer entries on the SpCas9-NG row, which inherits SpCas9 geometry.
- 2Engineered CRISPR-Cas9 nuclease with expanded targeting spaceNishimasu H, Shi X, Ishiguro S, et al. (Nureki O, Zhang F) · Science 361(6408):1259-1262; doi:10.1126/science.aas9129; PMID 30166441 · 2018The SpCas9-NG row's PAM = NG and the "Engineered SpCas9 variant" organism cell, plus the note "relaxed PAM requirement". Verified: the paper reports "a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs", with editing at NGA/NGT/NGG and lower activity at NGC. CAVEAT: this paper does NOT itself restate the 20 nt spacer or the ~3 bp cut position for the variant — those two cells on that row rest on Jinek 2012 above.
- 3In vivo genome editing using Staphylococcus aureus Cas9Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, et al. (Zhang F) · Nature 520(7546):186-191; doi:10.1038/nature14299; PMID 25830891 · 2015The whole SaCas9 row. Verified in full text: "SaCas9 cleaves genomic targets most efficiently with NNGRRT, all NNGRR PAMs can be cleaved and should be considered as potential targets" (backs PAM = NNGRRT, and shows the page's single string is the optimal case of a NNGRR family); and "SaCas9 achieves the highest editing efficiency in mammalian cells with guides between 21- to 23-nt long" (backs the 21 nt spacer — note the paper gives a 21-23 nt range, so the page's flat "21 nt" is the low end of that range, not the only length reported). Also backs the "smaller Cas9 ortholog / AAV delivery" note ("more than 1 kilobase shorter", packaged with its sgRNA cassette into a single AAV).
- 4Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas SystemZetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, et al. (Zhang F) · Cell 163(3):759-771; doi:10.1016/j.cell.2015.09.038; PMID 26422227 · 2015The Cas12a/Cpf1 row's orientation, spacer length and cut rule. Verified in full text: "The PAM for FnCpf1 is located upstream of the 5' end of the displaced strand of the protospacer" (backs orientation = 5' of spacer); a staggered double-strand break producing "a 5-nt 5' overhang" distal to the seed (backs "Staggered cut distal to PAM" and the "sticky ends instead of blunt Cas9 cuts" note); "Each mature crRNA begins with 19 nt of the direct repeat followed by 23-25 nt of the spacer sequence" (backs the 23-24 nt spacer, which sits inside the paper's 23-25 nt range). NOTE: this paper reports the PAM only as 5'-TTN (FnCpf1), NOT as TTTV — the TTTV string is backed by Kleinstiver 2019 below.
- 5Nomenclature for Incompletely Specified Bases in Nucleic Acid Sequences (Recommendations 1984)Nomenclature Committee of the International Union of Biochemistry (NC-IUB) · NC-IUB / IUBMB; published Nucleic Acids Res 1985;13:3021-3030, Eur J Biochem 1985;150:1-5, Biochem J 1985;229:281-286 · 1984The entire "Reading PAM ambiguity codes" table (all three rows) and the sentence introducing it. Verified on the official IUBMB-hosted recommendation: R = "G or A" (puRine), V = "G or C or A" (not-T), N = "G or A or T or C" (aNy). This is the primary recommendation itself, not a secondary reproduction.