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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] 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] DescriptionDiscoveryCRISPR-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] ClassificationCRISPR-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] NamingThe 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] StructureThe 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 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]
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]
OriginCas12 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] ToolsMultiple 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 propertyCRISPR-Cas9 is subject to Intellectual property disputes while CRISPR-Cas12a does not have the same issues.[2] NotesReferences
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