Wikipedia

Cas12a

Cas12a (CRISPR-associated protein 12a, previously known as Cpf1) is an RNA-guided endonuclease that forms an essential component of the CRISPR systems found in some bacteria and archaea. In its natural context, Cas12a targets and destroys the genetic material of viruses and other foreign mobile genetic elements, thereby protecting the host cell from infection. Like other Cas enzymes, Cas12a binds to a "guide" RNA (termed a crRNA, or CRISPR RNA) which targets it to a DNA sequence 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 (e.g. a portion of a phage genome) that previously infected the cell.[1]

CRISPR-associated protein 12a
Acidaminococcus sp. Cas12a PDB: 5B43​
Identifiers
SymbolCas12a
InterProIPR027620

As with Cas9 and other Cas proteins, the programmable DNA-targeting activity of Cas12a makes it a useful tool for biotechnology and biological research applications. By modifying the spacer sequence in the crRNA, researchers can target Cas12a to specific DNA sequences, allowing for highly targeted modifications of DNA.[2] Cas12a is distinguished from Cas9 by a its single RuvC endonuclease active site, its 5' protospacer adjacent motif preference, and its formation of sticky rather than blunt ends at the cut site; these and other differences may make it more suitable for certain applications[citation needed]. Beyond its use in basic research, CRISPR-Cas12a could have applications in the treatment of genetic illnesses and in implementing gene drives.[2]

Description

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Discovery

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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]

Classification

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CRISPR-Cas systems are separated into two classes: Class I, in which several Cas proteins associate with a crRNA to build a functional endonuclease, and Class II, in which a single Cas endonuclease associates with a crRNA; Class II is further divided into Type II, Type V, and Type VI systems. Cas12a is identified as a Class II, Type V CRISPR-Cas system.[4]

Naming

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The acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) refers to the invariant DNA sequences found in bacteria and archaea which encode Cas proteins and their crRNAs. Cas12a was originally known as Cpf1, 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

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The Cas12a protein contains a mixed alpha/beta domain, a RuvC-like endonuclease domain (broken into two non-contiguous segments, RuvC-I and RuvC-II) similar to the RuvC domain of Cas9, and a zinc finger-like domain[5]. Unlike Cas9, Cas12a does not have an HNH endonuclease domain, and the N-terminal region of Cas12a does not have an alpha-helical recognition lobe as seen in Cas9.[4]

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]

Also unlike Cas9, Cas12a does not require a tracrRNA (which in natural CRISPR systems must base-pair with a separate crRNA before binding to a Cas protein), instead binding a single crRNA. Both Cas12a and its guide RNA are smaller than the protein and RNA components of the Cas9 system; the crRNA of Cas12a is approximately half as long as sgRNAs used with Cas9.[6] This reduced size renders Cas12a more suitable for applications such as in vivo delivery via adeno-associated virus (AAV), which have limited DNA packaging capacity due to their small capsids.

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

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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

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Cas12a and Cas9 nucleases and their DNA cleavage positions

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

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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

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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

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CRISPR-Cas9 is subject to Intellectual property disputes while CRISPR-Cas12a does not have the same issues.[2]

Notes

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References

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  1. ^ a b "CRISPR-Based Genetic Engineering Gets a Kick in the Cas". Meta Science News. 2015-09-29. Archived from the original on 2017-10-22. Retrieved 2016-05-03.
  2. ^ a b c d "Even CRISPR". The Economist. ISSN 0013-0613. Retrieved 2016-05-03.
  3. ^ a b c Ledford H (October 2015). "Bacteria yield new gene cutter". Nature. 526 (7571): 17. doi:10.1038/nature.2015.18432. PMID 26432219.
  4. ^ a b c d Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, et al. (November 2015). "An updated evolutionary classification of CRISPR-Cas systems". Nature Reviews. Microbiology. 13 (11): 722–736. doi:10.1038/nrmicro3569. PMC 5426118. PMID 26411297.
  5. ^ a b c Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. (October 2015). "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system". Cell. 163 (3): 759–771. doi:10.1016/j.cell.2015.09.038. PMC 4638220. PMID 26422227.
  6. ^ a b "Cpf1 Moves in on Cas9 for Next-Gen CRISPR Genome Editing". epigenie.com. 29 September 2015. Retrieved 2016-05-03.
  7. ^ Fonfara I, Richter H, Bratovič M, Le Rhun A, Charpentier E (April 2016). "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA". Nature. 532 (7600): 517–521. Bibcode:2016Natur.532..517F. doi:10.1038/nature17945. PMID 27096362. S2CID 2271552.
  8. ^ "Nucleotide Codes, Amino Acid Codes, and Genetic Codes". KEGG: Kyoto Encyclopedia of Genes and Genomes. July 15, 2014. Retrieved 2016-05-25.
  9. ^ Cofsky JC, Karandur D, Huang CJ, Witte IP, Kuriyan J, Doudna JA (June 2020). "CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks". eLife. 9. doi:10.7554/eLife.55143. PMC 7286691. PMID 32519675.
  10. ^ Yamano T, Nishimasu H, Zetsche B, Hirano H, Slaymaker IM, Li Y, et al. (May 2016). "Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA". Cell. 165 (4): 949–962. doi:10.1016/j.cell.2016.04.003. PMC 4899970. PMID 27114038.
  11. ^ Altae-Tran H, Kannan S, Demircioglu FE, Oshiro R, Nety SP, McKay LJ, et al. (October 2021). "The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases". Science. 374 (6563): 57–65. Bibcode:2021Sci...374...57A. doi:10.1126/science.abj6856. PMC 8929163. PMID 34591643.
  12. ^ Graham DB, Root DE (November 2015). "Resources for the design of CRISPR gene editing experiments". Genome Biology. 16: 260. doi:10.1186/s13059-015-0823-x. PMC 4661947. PMID 26612492.
  13. ^ "Anti-CPF1 antibody (GTX133301) | GeneTex". www.genetex.com. Retrieved 2020-10-30.
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