Cas12a (CRISPR-associated protein 12a, previously known as Cpf1) is a subtype of Cas12 proteins and an RNA-guided endonuclease that forms part of the CRISPR system in some bacteria and archaea. In CRISPR systems, Cas12a serves to destroy the genetic material of viruses and other foreign DNA, thereby protecting the cell from infection. Like other Cas enzymes, Cas12a binds to an RNA (termed a crRNA in the case of Cas12a) to target nucleic acid in a specific and programmable matter. In the host organism, the crRNA contains a constant region that is recognized by the Cas12a protein and a spacer region that is complementary to a piece of foreign nucleic acid (i.e., phage) that previously infected the cell.[1]
The programmable nature of Cas12a makes this enzyme a useful tool for biotechnology and biological research. By modifying the spacer sequence in the crRNA, researchers can target Cas12a to specific DNA sequences, allowing for the creation of highly targeted modifications of DNA, similar to CRISPR-Cas9 system. Cas12a is distinguished from Cas9 by a its single RuvC endonuclease active site, its 5' protospacer adjacent motif preference, and for creating sticky rather than blunt ends at the cut site. These and other differences may make it more suitable in certain applications. Beyond its use in basic research, CRISPR-Cas12a could have applications in the treatment of genetic illnesses and in implementing gene drives.[2]
CRISPR-Cas12a was found by searching a published database of bacterial genetic sequences for promising bits of DNA. Its identification through bioinformatics as a CRISPR system protein, its naming, and a hidden Markov model (HMM) for its detection were provided in 2012 in a release of the TIGRFAMs database of protein families. Cas12a appears in many bacterial species. The ultimate Cas12a endonuclease that was developed into a tool for genome editing was taken from one of the first 16 species known to harbor it.[3] Two candidate enzymes from Acidaminococcus and Lachnospiraceae display efficient genome-editing activity in human cells.
CRISPR-Cas systems are separated into two classes: Class 1 uses several Cas proteins together with the CRISPR RNAs (crRNA) to build a functional endonuclease, while Class 2 CRISPR systems use a single Cas endonuclease with a crRNA. Under this classification, Cas12a has been identified as a Class II, Type V CRISPR-Cas system.[4]
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is named for the features of the invariant DNA sequences involved in targeting. Cas12a was originally known as Cpf1 as an abbreviation of CRISPR and two genera of bacteria where it appears, Prevotella and Francisella. It was renamed in 2015 after a broader rationalization of the names of Cas (CRISPR associated) proteins to correspond to their sequence homology.
The Cas12a locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain.[5] The Cas12a protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cas12a does not have a HNH endonuclease domain, and the N-terminal of Cas12a does not have the alpha-helical recognition lobe of Cas9.
Cas12a CRISPR-Cas domain architecture shows that Cas12a is functionally unique, being classified as Class 2, type V CRISPR system. The Cas12a loci encode Cas1, Cas2 and Cas4 proteins more similar to types I and III than from type II systems. Database searches suggest the abundance of Cas12a-family proteins in many bacterial species.
Functional Cas12a doesn't need the tracrRNA, therefore, only crRNA is required. This benefits genome editing because Cas12a is not only smaller than Cas9, but also it has a smaller sgRNA molecule (proximately half as many nucleotides as Cas9).[6]
The Cas12a-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif (PAM) 5'-YTN-3'[7] (where "Y" is a pyrimidine[8] and "N" is any nucleobase), in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cas12a introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucleotides overhang.
The CRISPR-Cas12a system consist of a Cas12a enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA. CRISPR-Cas12a systems activity has three stages:
Cas9 requires two RNA molecules to cut DNA while Cas12a needs one. The proteins also cut DNA at different places, offering researchers more options when selecting an editing site. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind blunt ends. Cas12a leaves one strand longer than the other, creating sticky ends. The sticky ends have different properties than blunt ends during non-homologous end joining or homologous repair of DNA, which confers certain advantages to Cas12a when attempting gene insertions, compared to Cas9. Although the CRISPR-Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in. Cas12a lacks tracrRNA, utilizes a T-rich PAM and cleaves DNA via a staggered DNA DSB.
In summary, important differences between Cas12a and Cas9 systems are that Cas12a:[10]
| Feature | Cas9 | Cas12a | |
|---|---|---|---|
| Structure | Two RNA required (Or 1 fusion transcript (crRNA+tracrRNA=sgRNA) | One crRNA required | |
| Cutting mechanism | Blunt end cuts | Staggered end cuts | |
| Cutting site | Proximal to recognition site | Distal from recognition site | |
| Target sites | G-rich PAM | T-rich PAM |
Cas12 endonucleases ultimately likely evolved from the TnpB endonuclease of IS200/IS605-family transposons. TnpB, not yet "domesticated" into the CRISPR immune system, are themselves able to perform RNA-guided cleavage using a OmegaRNA template system.[11]
Multiple aspects influence target efficiency and specificity when using CRISPR, including guide RNA design. Many design models and CRISPR-Cas software tools for optimal design of guide RNA have been developed. These include SgRNA designer, CRISPR MultiTargeter, SSFinder.[12] In addition, commercial antibodies are available for use to detect Cas12a protein.[13]
CRISPR-Cas9 is subject to Intellectual property disputes while CRISPR-Cas12a does not have the same issues.