Cas12a (CRISPR associated protein 12a, previously known as Cpf1) is an RNA-guided

endonuclease Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...

of that forms part of the

CRISPR CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacte ...

system in some

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...

and is used by scientists to modify DNA. It originates as part of a bacterial immune mechanism, where it serves to destroy the genetic material of

viruses A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky's 1 ...

and thus protect the cell and colony from viral infection. Cas12a and other CRISPR associated endonucleases are unique in that their use of a

guide RNA A guide RNA (gRNA) is a piece of RNA that functions as a guide for RNA- or DNA-targeting enzymes, with which it forms complexes. Very often these enzymes will delete, insert or otherwise alter the targeted RNA or DNA. They occur naturally, serv ...

makes this action highly selective; they only cut DNA when it is adjacent to a certain sequence of nucleotides. In the organisms from which it originates, this guide RNA is a copy of a piece of the genome from a virus that previously infected the cell. 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

Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic e ...

system. Cas12a is distinguished from Cas9 by being smaller and simpler, not requiring a

tracrRNA In molecular biology, trans-activating crispr RNA (tracrRNA) is a small ''trans''-encoded RNA. It was first discovered by Emmanuelle Charpentier in her study of human pathogen ''Streptococcus pyogenes'', a type of bacteria that causes harm to human ...

, and 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 Basic research, also called pure research or fundamental research, is a type of scientific research with the aim of improving scientific theories for better understanding and prediction of natural or other phenomena. In contrast, applied resear ...

, CRISPR/Cas12a could have applications in the treatment of genetic illnesses and in implementing

gene drive A gene drive is a natural process and technology of genetic engineering that propagates a particular suite of genes throughout a population by altering the probability that a specific allele will be transmitted to offspring (instead of the Mende ...

Description

Discovery

CRISPR/Cas12a was found by searching a published database of bacterial genetic sequences for promising bits of DNA. Its identification through

bioinformatics Bioinformatics () is an interdisciplinary field that develops methods and software tools for understanding biological data, in particular when the data sets are large and complex. As an interdisciplinary field of science, bioinformatics combi ...

as a CRISPR system protein, its naming, and a

hidden Markov model A hidden Markov model (HMM) is a statistical Markov model in which the system being modeled is assumed to be a Markov process — call it X — with unobservable ("''hidden''") states. As part of the definition, HMM requires that there be an ob ...

(HMM) for its detection were provided in 2012 in a release of the

TIGRFAMs TIGRFAMs is a database of protein families designed to support manual and automated genome annotation. Each entry includes a multiple sequence alignment and hidden Markov model (HMM) built from the alignment. Sequences that score above the defined ...

database of

protein families A protein family is a group of evolutionarily related proteins. In many cases, a protein family has a corresponding gene family, in which each gene encodes a corresponding protein with a 1:1 relationship. The term "protein family" should not be ...

. 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 ''Acidaminococcus'' is a genus in the phylum Bacillota (Bacteria), whose members are anaerobic diplococci that can use amino acids as the sole energy source for growth. Like other members of the class Negativicutes, they are gram-negative, despi ...

'' and ''

Lachnospiraceae The Lachnospiraceae are a family of obligately anaerobic, variably spore-forming bacteria in the order Eubacteriales that ferment diverse plant polysaccharides to short-chain fatty acids (butyrate, acetate) and alcohols (ethanol). These bacteria ...

'' display efficient genome-editing activity in human cells. A smaller version of Cas9 from the bacterium ''

Staphylococcus aureus ''Staphylococcus aureus'' is a Gram-positive spherically shaped bacterium, a member of the Bacillota, and is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It is often positive ...

'' is a potential alternative to Cas12a.

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.Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas systems." Nature Reviews Microbiology (2015).

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 Genus ( plural genera ) is a taxonomic rank used in the biological classification of living and fossil organisms as well as viruses. In the hierarchy of biological classification, genus comes above species and below family. In binomial nomenclat ...

of bacteria where it appears,

Prevotella ''Prevotella'' is a genus of Gram-negative bacteria. ''Prevotella'' spp. are members of the oral, vaginal, and gut microbiota and are often recovered from anaerobic infections of the respiratory tract. These infections include aspiration pneum ...

and

Francisella ''Francisella'' is a genus of Gram-negative bacteria. They are small coccobacillary or rod-shaped, nonmotile organisms, which are also facultative intracellular parasites of macrophages. Strict aerobes, ''Francisella'' colonies bear a morpholog ...

. It was renamed in 2015 after a broader rationalization of the names of Cas (CRISPR associated) proteins to correspond to their

sequence homology Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a spe ...

Structure

The Cas12a locus contains a mixed

alpha Alpha (uppercase , lowercase ; grc, ἄλφα, ''álpha'', or ell, άλφα, álfa) is the first letter of the Greek alphabet. In the system of Greek numerals, it has a value of one. Alpha is derived from the Phoenician letter aleph , whic ...

beta Beta (, ; uppercase , lowercase , or cursive ; grc, βῆτα, bē̂ta or ell, βήτα, víta) is the second letter of the Greek alphabet. In the system of Greek numerals, it has a value of 2. In Modern Greek, it represents the voiced labiod ...

domain, a RuvC-I followed by a helical region, a RuvC-II and a

zinc finger A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) in order to stabilize the fold. It was originally coined to describe the finger-like appearance of a hypothesized struct ...

-like domain. 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 CRISPR-associated protein 1 (cas1) is one of the two universally conserved proteins found in the CRISPR prokaryotic immune defense system. Cas1 is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments. Cas1 forms ...

, 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

, 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

nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...

s as Cas9). The Cas12a-crRNA complex cleaves target DNA or RNA by identification of a

protospacer adjacent motif A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of the invading virus or plasmid, but ...

(PAM) 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any

nucleobase Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...

), 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.

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: * 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 Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

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 DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the ...

. Cas12a leaves one strand longer than the other, creating

sticky ends DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the ...

. The sticky ends have different properties than blunt ends during

non-homologous end joining Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology direct ...

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

, 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 Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology direct ...

or HDR. * Cuts target DNA further away from PAM, further away from the Cas9 cutting site, enabling new possibilities for cleaving the DNA.

Origin

Both Cas9 and Cas12 endonucleases ultimately originates 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.

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. In addition, commercial antibodies are available for use to detect Cas12a protein.

Intellectual property

CRISPR/Cas9 is subject to

Intellectual property Intellectual property (IP) is a category of property that includes intangible creations of the human intellect. There are many types of intellectual property, and some countries recognize more than others. The best-known types are patents, cop ...

disputes while CRISPR/Cas12a does not have the same issues.

Notes

References

{{DEFAULTSORT:CRISPR Cpf1 Genetic engineering Enzymes Genome editing