Biology:Cas12a
| CRISPR-associated protein 12a | |
|---|---|
 | |
| Identifiers | |
| Symbol | Cas12a |
| InterPro | IPR027620 |
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. It originates as part of a bacterial immune mechanism, where it serves to destroy the genetic material of viruses and thus protect the cell and colony from viral infection. Cas12a and other CRISPR associated endonucleases use an RNA (termed a crRNA in the case of Cas12a) to target nucleic acid in a specific and programmable matter. In the organisms from which it originates, this guide RNA is a copy of a piece of foreign nucleic acid (i.e. phage) that previously infected the cell.[1]
It is of interest to researchers because it can be used to make highly targeted modifications of DNA or RNA, similar to the better known CRISPR-Cas9 system.[2] 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]
Description
Discovery
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.[2]
A smaller version of Cas9 from the bacterium Staphylococcus aureus is a potential alternative to Cas12a.[3]
Classification
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 only a single Cas protein with a crRNA. Under this classification, Cas12a has been identified as a Class II, Type V CRISPR-Cas system containing a 1,300 amino acid protein.[4]
Name
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.[4]
Structure
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.[4]
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.[4]
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.[5]
Mechanism
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:[3]
- Adaptation: Cas1 and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array.
- Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein.
- Interference: the Cas12a is bound to a crRNA to form a binary complex to identify and cleave a target DNA sequence. The crRNA-Cas12a complex searches dsDNA for a 3-6nt 5' protospacer adjacent motif (PAM). Once a PAM is found, the protein locally denatures the dsDNA and searches for complementarity between the crRNA spacer and the ssDNA protospacer. Sufficient complementarity will trigger RuvC activity and the RuvC active site will then cut the non-target strand and then the target strand, ultimately generating a staggered dsDNA break with 5' ssDNA overhangs (cis cleavage).[5][9]
Cas9 vs. Cas12a
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.[3] Although the CRISPR-Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in.[1] Cas12a lacks tracrRNA, utilizes a T-rich PAM and cleaves DNA via a staggered DNA DSB.[6]
In summary, important differences between Cas12a and Cas9 systems are that Cas12a:[10]
- Recognizes different PAMs, enabling new targeting possibilities.
- Creates 4-5 nt long sticky ends, instead of blunt ends produced by Cas9, enhancing the efficiency of genetic insertions and specificity during NHEJ or HDR.
- Cuts target DNA further away from PAM, further away from the Cas9 cutting site, enabling new possibilities for cleaving the DNA.
| 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 |
Origin
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]
Tools
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]
Intellectual property
CRISPR-Cas9 is subject to Intellectual property disputes while CRISPR-Cas12a does not have the same issues.[2]
Notes
References
- ↑ 1.0 1.1 "CRISPR-Based Genetic Engineering Gets a Kick in the Cas" (in en-US). 2015-09-29. http://news.meta.com/?p=863.
- ↑ 2.0 2.1 2.2 2.3 "Even CRISPR". The Economist. ISSN 0013-0613. https://www.economist.com/news/science-and-technology/21668031-scientists-have-found-yet-another-way-edit-genomes-suggesting-such-technology-will.
- ↑ 3.0 3.1 3.2 3.3 "Bacteria yield new gene cutter". Nature 526 (7571): 17. October 2015. doi:10.1038/nature.2015.18432. PMID 26432219.
- ↑ 4.0 4.1 4.2 4.3 "An updated evolutionary classification of CRISPR-Cas systems". Nature Reviews. Microbiology 13 (11): 722–736. November 2015. doi:10.1038/nrmicro3569. PMID 26411297.
- ↑ 5.0 5.1 5.2 "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system". Cell 163 (3): 759–771. October 2015. doi:10.1016/j.cell.2015.09.038. PMID 26422227.
- ↑ 6.0 6.1 "Cpf1 Moves in on Cas9 for Next-Gen CRISPR Genome Editing". 29 September 2015. http://epigenie.com/cpf1-takes-crispr-bigger-by-going-smaller/.
- ↑ "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA". Nature 532 (7600): 517–521. April 2016. doi:10.1038/nature17945. PMID 27096362. Bibcode: 2016Natur.532..517F.
- ↑ "Nucleotide Codes, Amino Acid Codes, and Genetic Codes". KEGG: Kyoto Encyclopedia of Genes and Genomes. July 15, 2014. http://www.genome.jp/kegg/catalog/codes1.html.
- ↑ "CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks". eLife 9. June 2020. doi:10.7554/eLife.55143. PMID 32519675.
- ↑ "Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA". Cell 165 (4): 949–962. May 2016. doi:10.1016/j.cell.2016.04.003. PMID 27114038.
- ↑ "The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases". Science 374 (6563): 57–65. October 2021. doi:10.1126/science.abj6856. PMID 34591643. Bibcode: 2021Sci...374...57A.
- ↑ "Resources for the design of CRISPR gene editing experiments". Genome Biology 16: 260. November 2015. doi:10.1186/s13059-015-0823-x. PMID 26612492.
- ↑ "Anti-CPF1 antibody (GTX133301) | GeneTex". https://www.genetex.com/Product/Detail/CPF1-antibody/GTX133301.
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