Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cas12a (previously termed Cpf1) is a DNA-editing technology analogous to the CRISPR/Cas9 system. Cas12a is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cas12a genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cas12a is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. CRISPR/Cas12a could have multiple applications, including treatment of genetic illnesses and degenerative conditions.
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. 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. Class 2 CRISPR systems use a single Cas protein with a crRNA. Cpf1 has been recently identified as a Class II, Type V CRISPR/Cas systems containing a 1,300 amino acid protein.
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. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have a HNH endonuclease domain, and the N-terminal of Cas12a does not have the alpha-helical recognition lobe of Cas9.
Cpf1 CRISPR-Cas domain architecture shows that Cpf1 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 Cpf1-family proteins in many bacterial species.
Functional Cpf1 doesn’t need the tracrRNA, therefore, only crRNA is required. This benefits genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (proximately half as many nucleotides as Cas9).
The Cas12a-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5'-YTN-3' (where "Y" is a pyrimidine 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/Cpf1 systems activity has three stages:
- 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.
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 aid in the incorporation of new sequences of DNA, making Cas12a more efficient at gene introductions than 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:
- 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.
|Structure||Two RNA required (Or 1 fusion transcript (crRNA+tracrRNA=gRNA)||One RNA 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|
Multiple aspects influence target efficiency and specificity when using CRISPR, including guide RNA design. Many design models for guide RNA have been suggested, with tools to facilitate optimized design. These include SgRNA designer, CRISPR MultiTargeter, SSFinder. In addition, commercial antibodies are available for use to detect Cpf1 protein.
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