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Short answer
Cas12a, first described as Cpf1, is a CRISPR-associated nuclease that is guided by a single CRISPR RNA (crRNA), needs no tracrRNA, recognizes a T-rich protospacer adjacent motif (PAM) on double-stranded DNA, and cuts DNA with a staggered break. Once activated it can also cut nearby single-stranded DNA that does not match the guide.
Definition and scope
Cas12a is an RNA-guided endonuclease. The 2015 paper that characterized it in Cell summarized it this way: “Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer adjacent motif.” The same paper tested 16 Cpf1-family proteins and found that two, from Acidaminococcus (AsCpf1) and Lachnospiraceae (LbCpf1), edited efficiently in human cells. The enzyme is now usually called Cas12a, so AsCas12a and LbCas12a are the same two proteins under the current name.
This page explains the mechanism and the vocabulary. It is not a protocol and does not cover experimental design for a particular cell type or organism. For the wider picture of what CRISPR tools can and cannot do, see CRISPR pros and cons.
How Cas12a works in practice
One RNA guides it
Cas9 systems use a crRNA together with a tracrRNA. Cas12a does not need the tracrRNA, so the guide is a single, shorter RNA. In Zetsche et al., the mature crRNA is 42-44 nucleotides, made of a 19-nucleotide direct repeat followed by a 23-25 nucleotide spacer that pairs with the target.
A T-rich PAM and a staggered cut
For double-stranded DNA targets, Cas12a requires a PAM. The paper states the motif as 5′-TTN (and also reports targeting of 5′-CTA). The paper states that “Cpf1 cleaves DNA via a staggered DNA double stranded break”, and reports that FnCpf1-mediated cleavage leaves a 5-nucleotide 5′ overhang.
Cis and trans cleavage
This is the part that most overview pages skip, and it is the reason Cas12a is discussed outside genome editing at all. Li et al. (Cell Research, 2018) showed that Cas12a has two activities on single-stranded DNA (ssDNA):
- Cis-cleavage: programmable on-target cleavage of the target ssDNA that the crRNA pairs with.
- Trans-cleavage: once the crRNA, Cas12a and target form a ternary complex, the complex “promiscuously cleaved collateral ssDNAs that had no complementarity to the crRNA guide sequence.”
Two further details from that paper matter when reading methods sections. First, for ssDNA targets “the cleavage did not require the existence of a PAM sequence”; the T-rich PAM requirement described above belongs to double-stranded targets. Second, both activities come from the same RuvC catalytic pocket: in FnCas12a, the substitutions D917A, E1006A and D1255A completely abolished or markedly decreased ssDNA cleavage. The authors note that the trans activity “may also be utilized in potential biotechnological applications”, by analogy with Cas13a-based detection.
Cas12a, Cas9 and Cas13 side by side
A short comparison of the points covered in the two papers cited below and in the CASRAI Cas13 guide.
| Question | Cas12a | Cas9 and Cas13 |
|---|---|---|
| Guide RNA components | crRNA only; no tracrRNA | Cas9 systems use crRNA plus tracrRNA |
| Typical target | DNA (double-stranded with a T-rich PAM; single-stranded without a PAM requirement) | Cas13 targets RNA; see Cas13 and RNA targeting |
| Cut geometry on dsDNA | Staggered, 5-nucleotide 5′ overhang | Not covered here |
| Collateral cleavage after target binding | Reported for ssDNA (trans-cleavage) | Li et al. draw an analogy to Cas13a |
What to record in a methods section
When reading or writing a Cas12a methods section, these details help others reproduce the work. The checklist is built from the two papers cited below.
- Which ortholog: AsCas12a (Acidaminococcus) or LbCas12a (Lachnospiraceae), since these were the two efficient in human cells.
- Guide format: a single crRNA, with its length stated (the mature form in the original paper was 42-44 nucleotides).
- PAM context: the T-rich PAM at each double-stranded target site (5′-TTN in the original paper).
- Target type: double-stranded or single-stranded DNA, because PAM dependence differs.
- Catalytic variants: any RuvC substitutions used, such as D917A, E1006A or D1255A.
Work with recombinant DNA and gene-editing reagents usually falls under institutional oversight; see what an Institutional Biosafety Committee does, and transfection methods and biosafety approval for how delivery choices connect to that review. If you report quantitative PCR results alongside editing outcomes, the MIQE reporting checklist covers what to document.
Common misunderstandings
- “Cas12a and Cas9 are interchangeable.” The original paper describes a single-crRNA enzyme with a T-rich PAM, so check the guide format and PAM requirement of the specific enzyme before reusing a design.
- “A PAM is always required.” Li et al. report that ssDNA cleavage did not require a PAM; the PAM requirement applies to double-stranded targets.
- “Trans-cleavage makes Cas12a a validated diagnostic.” The cited paper shows the activity and says it may be useful for biotechnology. It does not describe a diagnostic assay, so a detection claim needs its own validation data.
- “Cpf1 and Cas12a are different enzymes.” They are the same family; Cpf1 is the earlier name used in the 2015 paper.
- “Cas12a cuts RNA.” The activities described here are on DNA. RNA-targeting systems such as Cas13 are covered separately.
Frequently asked questions
What is Cas12a?
Cas12a, originally called Cpf1, is a single RNA-guided endonuclease that lacks a tracrRNA and uses a T-rich PAM (Zetsche et al., 2015).
Is Cas12a the same as Cpf1?
Yes. Cpf1 is the earlier name used in the 2015 Cell paper; the enzyme is now usually called Cas12a.
Does Cas12a need a PAM?
For double-stranded DNA targets it does (5′-TTN in the original paper). Li et al. report that cleavage of single-stranded DNA targets did not require a PAM.
What is trans-cleavage?
After the crRNA, Cas12a and target form a ternary complex, the complex also cleaves collateral single-stranded DNA with no complementarity to the guide (Li et al., 2018).
Does Cas12a cut RNA?
The activities described in the two papers are on DNA. RNA targeting is covered in the Cas13 guide.
Sources
- Zetsche B, et al. Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell 2015;163:759-771. doi 10.1016/j.cell.2015.09.038. PubMed Central full text, checked 2026-10-29.
- Li S-Y, et al. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Research 2018;28(4):491-493. doi 10.1038/s41422-018-0022-x. PubMed Central full text, checked 2026-10-29.
