Cas9 and Cas12a compared

What is the difference between Cas9 and Cas12a?

Both are RNA-guided CRISPR enzymes that cut both DNA strands. Cas12a, also called Cpf1, recognises a T-rich signal next to its target, uses a single short RNA and leaves a staggered cut, whereas Cas9 needs a different signal and a longer guide.

Cas9

Cas9 was shown in 2012 to be programmable with a single guide RNA to cut a chosen DNA target ^[1]. It needs a short signal next to the target, called a PAM, and each of its two cutting domains cuts one strand ^[1].

Cas12a

Cas12a was described in 2015 as a distinct single RNA-guided enzyme ^[6]. It recognises a T-rich PAM, needs no second RNA, and works with a single CRISPR RNA only 42 to 44 letters long ^[6]. It cuts both strands at staggered points, leaving a short overhang rather than blunt ends ^[6][7].

Cas12a can also process its own precursor RNA into mature guides ^[7]. That means several guides can be made from one transcript.

Side by side

The published descriptions of the two enzymes differ on four points ^[1][6][7].

• Target signal: Cas9 and Cas12a read different PAMs, so each reaches sites the other cannot ^[1][6].
• Guide: Cas9 uses a single guide RNA made by joining two natural RNAs; Cas12a uses one short CRISPR RNA ^[1][6].
• Cut: Cas12a leaves a staggered cut with an overhang ^[6][7].
• Guide processing: Cas12a can cut its own precursor RNA into several guides ^[7].

Use in plants

Both enzymes have published records in crops. Custom guide RNAs directed Cas9 to make targeted changes in rice and common wheat in 2013 ^[8]. In 2017, one form of Cas12a produced changes in both copies of the target at nearly 100% efficiency at four sites in first-generation transgenic rice plants ^[9]. In the same study, Cas12a forms were repurposed to switch down a gene in Arabidopsis rather than cut it ^[9].

Where they sit among the five methods

Cas9 and Cas12a are both CRISPR nucleases. The other methods covered here work differently: TALENs and zinc finger nucleases find their target with custom proteins rather than an RNA guide, while base editing and prime editing change the sequence without cutting both strands ^[2][3][4][5].

Frequently asked questions

Is Cpf1 the same as Cas12a?

Yes. Cpf1 was the name used when the enzyme was described in 2015; it is now more widely known as Cas12a.

Is Cas12a more precise than Cas9?

The papers cited here describe how the two recognise and cut DNA, not a head-to-head ranking. Precision depends on the enzyme, the guide and the target chosen.

Why would a researcher choose one over the other?

One consideration is reach. Each enzyme needs its own signal next to the target, so the sequence around a gene affects which one can be pointed at it.

References

1. Science (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity (Jinek and others)
2. Genetics, via PubMed (2010). Targeting DNA double-strand breaks with TAL effector nucleases (Christian and others)
3. Proceedings of the National Academy of Sciences (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain (Kim, Cha and Chandrasegaran)
4. Nature (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage (Komor and others)
5. Nature (2019). Search-and-replace genome editing without double-strand breaks or donor DNA (Anzalone and others)
6. Cell (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system (Zetsche and others)
7. Nature (2016). The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA (Fonfara and others)
8. Nature Biotechnology (2013). Targeted genome modification of crop plants using a CRISPR-Cas system (Shan and others)
9. Nature Plants (2017). A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants (Tang and others)