CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic sequence and CRISPR-associated protein 9) has created a buzz in the Science world. The ability of this genome editing tool has enabled medical scientists and biological researchers worldwide to edit specific DNA sequences of the genome to serve many in vivo and in-vitro applications.
Based on their structure CRISPR/Cas is divided into two subclasses:
Class II: They mediate the interference through single or multi-domain effectors.
Based on the genomic architecture of the CRISPR array and the signature interference effector, these two classes are further subdivided into six types and 33 subtypes.
For example, Class I includes types I, III, and IV, and class II includes types II, V, and VI CRISPR/Cas systems. Type I, II, and V CRISPR/Cas systems target the double-stranded DNA of invading phages or conjugative plasmids. However, CRISPR/Cas systems of types III and VI target the single-stranded RNA of invading phages. This offers molecular immunity to organisms against invading nucleic acids.
CRISPR enzymes such as Cas9 and Cas12a (formerly Cpf1) have opened the door for manipulating and studying RNA in whatever way researchers want. During recent computational efforts to identify new CRISPR systems, Cas13 was discovered as a novel type of RNA targeting enzyme.
CRISPR-associated nuclease Cas13a, also known as C2C2, has single-stranded RNA and collateral RNA cleavage activities or trans-cleavage of mRNA transcripts. It was originally discovered in Leptotrichia shahii (also referred to as LshCas13a).
The enzyme consists of two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains, having the ability to differentiate between the CRISPR RNA and the single-stranded RNA to be edited.
In this article, we will teach you how Cas13a works in a CRISPR-Cas13a system and its applications in a spectrum of biotechnology, diagnostic, and research workflows.
CRISPR/Cas adaptive immunity systems impart resistance against invading pathogens and conjugative plasmids in bacteria and archaea. The immunity mechanism has three steps:
Cas13a facilitates immunity mechanisms through its two separate RNase activities: RNA detection or recognition and RNA cleavage. The catalytic sites responsible for these activities include the REC lobe (Having a Helical-1 domain) and the NUC lobe (with 2 HEPN domains).
In response to the formation of guide-target RNA duplexes, Cas13a is activated by triggering the HEPN1 domain, which then moves toward the HEPN2 domain to bind and cleave complementary target RNA. This non-specific RNase activity performed by Cas13a is known as the collateral effect.
The property is extensively exploited in labs for nucleic acid detection. One of the most commonly used nucleic acid detection assays involving a variant of Cas13a from Leptotrichia wadei is Specific High-Sensitivity Enzymatic Reporter unLOCKing (SHERLOCK).
The team East-Seletsky et al have shown in their studies that Cas13a has two different functions. One is the degradation of ssRNA targets and the second is the processing of the pre-crRNA. These activities facilitate the design and expression of multiple crRNAs under polymerase II promoter control for multiple transcription targets.
For ribonuclease activity, Cas13a requires a flexible sequence. The protospacer-flanking site (PFS) must consist of 3’–H where H can be uracil, adenine, or cytosine followed by the spacer sequence.
For example, LshCas13a requires a 64-nucleotide guide RNA for a functioning ribonucleoprotein. The guide RNA consists of a unique scaffold and a 22-30 nucleotide long spacer peculiar to the target sequence or target RNA of interest.
According to Gootenberg’s study (PMID: 28408723), the scaffold should be placed at the 5’ end and the sequence-specific guide on the 3’ end [5’–Scaffold + Guide–3′] of a reverse vector primer, while designing single guide RNAs for Cas13a. It allows the proper formation of the ribonucleoprotein complex.
CRISPR-Cas13a provides defense in organisms against viral forms. In bacterial cells, it’s used to bind and cleave RNA non-specifically.
Cas13a also has many in vivo and in vitro applications. It’s used to isolate specific RNA sequences from a population and study their processing in live cells. Several groups have used Cas13a fusion protein for modulating, imaging, splicing, tracking, and controlling targeted RNA expression.
In Gootneberg’s study, Cas13a has been used to specifically detect single RNA molecules. The system has been called SHERLOCK. Its practical applications include genotyping of humans, differentiating strains of the Zika virus, detecting COVID-19, and identifying tumor mutations within cell-free genomic DNA.
Studies have shown that Cas13a is one of the most effective systems for mammalian cell RNA binding and knockdown, compared to its orthologs found in E. coli.
The CRISPR/Cas system confers immunity to bacteria and archaea against invading nucleic acids and phages. The CRISPR/Cas13a (Formerly known as C2c2) refers to a class 2 type VI-A ribonuclease that targets single-stranded RNA molecules in the phage genome and cleaves them.
With its trans-cleavage activity, CRISPR-Cas13a serves as a powerful diagnostic tool. The SHERLOCK or DETECTOR system has detected many viruses, including COVID-19 causing SARS-Cov2, with single-base resolution and higher sensitivity.
Some researchers detected different viruses by using five different sets of virus-specific primers with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) in a single tube.
CRISPR/Cas13a system has a spectrum of applications in a range of life sciences, pharmaceutical, and biomedical labs. It’s an efficient tool for RNA detection and regulation of gene expression. It has extensive application in CRISPR-based genome editing and PCR amplification.
Furthermore, Abudayyeh and Cox’s studies show that the molecular tool works perfectly to cleave and edit target sequences in both mammalian and prokaryotic cells and detect nucleic acids with high sensitivity.
The collateral cleavage activity of Cas13a can be used to trigger programmed cell death to detect a specific RNA in vivo. Further, it can also identify specific RNA in vitro by degrading labeled RNA non-specifically.
Cas13a is an integral part of the RNA-guided RNA-targeting CRISPR/Cas system that acts as an effective diagnostic and research tool. It’s used to detect nucleic acids with single-base mismatch specificity and tumor-derived DNA and RNA in the blood. It has major applications in diagnosing and monitoring cancer and in its therapy guidance.
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CRISPR-Cas13 is an RNA-guided RNA endonuclease from Leptotrichia shahii. The Cas13a enzyme is involved in the cleavage of ssRNA and crRNA processing. Naturally, the enzyme has a role in providing defense to bacteria against invading nucleic acids or phages.
The collateral RNA cleavage activity of the enzyme is exploited in a range of biotechnology and molecular biology areas for the detection of specific RNA and sequence-specific cleavage. It has been tested in a range of cancer studies to inhibit tumor growth and angiogenesis in an intracranial glioma tumor model.
These studies involve high-throughput workflows such as multiplex PCR, cloning, transformation, immunoblotting, and others. These procedures need to be paired with high-tech equipment to obtain reliable and accurate data.
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