Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems have revolutionized biotech and medical fields with their unique gene manipulation mechanism.
The CRISPR pathway is part of bacteria's immune system that functions against viruses and plasmid DNA. When a virus invades bacteria, a short fragment of their genome is incorporated within the bacterial genome at CRISPR loci. It served as a memory for the bacteria to kill the virus when it invades in the future.
Re-infection by the same virus triggers complementary mature CRISPR RNA (crRNA) and a matching sequence is identified. Then, the Cas9 endonuclease locates the target sequence and causes the double-strand break. It functions as a self-programmable restriction enzyme.
Today, the CRISPR-Cas system is a versatile and effective tool for genome editing at a target sequence in a variety of organisms, from bacteria to humans.
Though many other high-throughput genome engineering techniques are available, such as transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs), the RNA-guided CRISPR-Cas system is the most widely used technology worldwide. It’s mainly because of its high efficiency, simple design, and short cycle.
CRISPR-Cas system is classified into two classes based on the number of effector proteins and cleavage of foreign nucleic acid:
Figure: CRISPR-Cas system and its classification.
Among all the types, the CRISPR-Cas9 system, found in Streptococcus pyogenes (SpCas9), is one of the widely used CRISPR systems in labs. The system is composed of two main elements, RNA-guided Cas9 endonuclease enzyme and single-guide RNA (sgRNA).
Cas9 is a protein with a molecular weight of 160 kilodaltons. It’s mainly involved in DNA cleavage and altering organisms’ genomes. That’s why it’s heavily utilized in genetic engineering and brought a Nobel prize to Emmanuelle Charpentier and Jennifer Doudna in 2020 for the development of the technology.
In this article, we will further review the working mechanisms of Cas9 and its variants, its functions, and applications in diverse fields.
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The Cas9 endonuclease consists of two domains: HNH and RuvC domain. Each domain is involved in the cleavage of the single strand of the target double-strand DNA of the organism. However, Cas9 does not do it alone. It requires single-guide RNA (sgRNA), which is a combination of crRNA and tracrRNA. The crRNA targets the cleavage of double-stranded DNA and contains a binding site for the tracrRNA, which has a stem-loop structure for the binding of Cas9 protein.
The csgRNA and Cas9 protein bind together to form a Cas9 ribonucleoprotein (RNP) that carries out the cleavage processes.
To bind and cut the target DNA at a specific location, RNP needs a protospacer (17–21 bases of RNA-to-DNA homology) and a protospacer adjacent motif (PAM) sequence, which is composed of the three-nucleotide sequence- NGG. The complex of Cas9. CRISPR RNA (crRNA), and trans-activating crRNA (tracrRNA) then recognize the target sequence, bind to them, and carry out the DNA cleavage process.
The presence of the nucleotide sequence, NGG, at the 3’ end of the target DNA is necessary for Cas9 to recognize and cleave the target DNA. In addition to NGG, some other PAM sequences, such as NGA and NAG, are also found. However, they are only found in about 16 percent of the human genome, and their efficiency for genome editing is low. This restricts genomic loci that can be targeted.
Several variants of Cas9 proteins are developed to expand the compatibility of the PAM sequence and enhance applications in different areas. Some Cas9 variants developed so far are:
Other than these, researchers have also developed a dCas9 variant by introducing two-point mutations, H840A and D10A, in Cas9 endonuclease. They can effectively bind DNA, but their DNA cleavage activity is compromised. Often, commercially available recombinant Cas9 nucleases (purified from E. coli strains) contain nuclear localization signals (NLS) for more efficient gene editing.
The unique DNA cleavage capability of the CRISPR-Cas9 system makes them desirable in labs and industries for genetic engineering applications. Out of many Cas9 systems available, wild-type streptococcus pyogenes Cas9 is a popular choice for CRISPR-based genome editing experiments.
Altering the genomic sequences of organisms is one of the common applications. The sgRNA guides Cas9 endonuclease to the target location of the target DNA and causes a double-stranded break (DSB). The DNA break is repaired using two techniques: the homology-directed repair (HDR) pathway or the error-prone non-homologous end joining (NHEJ) pathway.
Though the NHEJ is more efficient than HDR, it causes frameshift mutations or the inactivation of genes by forming stop codons within their open reading frame (ORF) by inserting insertions or deletions into the cleavage sites. The HDR mechanism is dependent on the homology donor and enables accurate modification of the target site within an organism’s genome.
However, the major limitation associated with using a CRISPR-Cas system for gene editing is alterations at off-target sites than the target site, causing hindrance in the generation of desired mutations. The effect can be reduced by injecting purified Cas9 protein in the target cell or by the recombinant protein.
Another approach is the use of a double-nicking strategy that employs a pair of sgRNAs and two mutant Cas9-D10A nickases to form a functional double-strand break at the target site of the DNA.
Figure: Schematic representation of CRISPR-Cas9 and its mutated form dCas9 in gene expression.
CRISPR-Cas9 is a versatile and essential tool in labs and industries for gene editing purposes. Its simple design and potential of cutting the target DNA with specificity make it a popular genetic engineering technique worldwide.
Today, many readymade CRISPR-Cas kits are available that contain transfection tools and all the other reagents required to perform genome editing experiments. However, often researchers prefer to curate their own set of reagents based on the purpose of their applications.
In biomedical labs, CRISPR-Cas is widely used to detect variant pathogens and study various critical diseases, such as cancer, genetic disorders, and virus infection. For example, it has been utilized to destroy some human infecting viruses human papillomaviruses, and HIV-1 provirus.
The tool has also shown great potential in cancer treatment and targeting human hereditary liver diseases.
The effective treatment of diseases depends on the target specificity and editing efficiency of the CRISPR-Cas9 system. That’s why researchers are now designing many recombinant proteins and CRISPR-Cas systems for their targeted applications. However, ethical issues, effective delivery systems, and immunogenicity remain to be the hindrance in the use of the system in clinical applications.
In agriculture, the CRISPR-Cas system is used to develop crops with improved nutrition and disease-resistant characteristics. Moreover, it increases crops' tolerance to drought and extends their shelf life.
The technique is also used in research to understand the functional roles of genes in diseases and infections. They are used to induce targeted deletion or insertions (indels) at the target site for gene modification. These sites can be detected using techniques such as mismatch assay (CEL-I).
The pathways or repair systems that CRISPR-Cas systems mainly use to modify DNA through mutagenesis at specific locations are HDR (add new DNA sequence) and NHEJ (knockout gene).
CRISPR-Cas9 holds great promise in treating genetic disorders, such as β-thalassemia, sickle cell disease, and muscular dystrophy. That’s why it’s a popular choice in gene therapy treatments. Gene therapy involves replacing malfunctioned or erroneous genes using exogenous DNA.
CRISPR-Cas system is also used in developing animal models for human diseases. Studies have shown that editing genes with CRISPR-Cas is more effective in lab settings (in vitro) compared to inside living organisms (in vivo). Because of this, scientists often use genetically modified cell models to speed up medical research. Until now, researchers have used CRISPR-Cas to make changes to genes in different cell lines, like tumor cells, adult cells, and stem cells, to mimic various human diseases and study them.
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Cas9 is an RNA-guided endonuclease enzyme of 160 kDa. With CRISPR, the enzyme forms the CRISPR-Cas9 system involved in genome editing and genetic engineering applications. CRISPR-Cas9 employs gRNA that directs the Cas9 protein to its target DNA sequence for cleavage.
It’s a widely used and versatile tool used in labs to understand gene functions or treat critical diseases, such as cancer and other genetic disorders. However, conducting such high-throughput applications demands the use of high-quality reagents and state-of-the-art equipment.
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