What is CRISPR-Cas9?

To understand CRISPR-Cas9, we must first get familiar with gene editing. Gene editing is the process of changing an organism’s genetic code or genome. Also referred to as genome editing, this study has a rich history of yielding and developing adjunct techniques in search of answers to life-threatening human diseases.

Several programmable sequence-specific endonucleases have been introduced to edit DNA and interrogate genetic elements that cause variations or diseases in humans, including Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs).

The most recent and compelling genome editing tool among these is CRISPR-Cas9. CRISPR stands for “clustered regularly interspaced short palindromic repeats,” a small stretch of a DNA sequence. Cas9 is CRISPR-associated protein 9.

The unique CRISPR-Cas system is a sensation in scientific communities because of its ability to cut a specific piece of DNA, alter it in the desired way, and modify gene function.

The CRISPR-Cas tool was first realized by Jennifer A. Doudna, Emmanuelle Charpentier, and Martin Jinek in 2012. They utilized CRISPR-Cas9 gene editing technology and discovered its ability to cut any genome at any desired place. Charpentier and Doudna later earned the 2020 Nobel Prize in Chemistry for their pioneering work.

In this article, we will cover the CRISPR-Cas9 system, its mechanisms, applications, and limitations.

How Does CRISPR Work?

The CRISPR-Cas9 system was adapted from the immune system of bacteria and archaea. They use six different types of CRISPR-Cas systems to protect themselves from invading viruses or any foreign agents. This was first demonstrated by Rodolphe Barrangou and a team of researchers in Streptococcus thermophilus bacteria.

Below is a high-level sequence of their defense mechanism:

1. Bacteriophage invades the bacterial cell.
2. Cas1 and Cas2 proteins acquire a piece of the bacteriophage’s DNA and integrate it into the CRISPR array.
3. The DNA is then transcribed into pre-crRNA.
4. The pre-crRNA is further processed and classified into crRNA by Type I, Type II, Type III, Type IV, Type V, or Type VI systems that are further involved in target localization and degradation.

Image: An illustration of the working mechanism of the CRISPR-Cas system in bacteria against bacteriophages.

CRISPR-Cas9

In most labs, the CRISPR-CAS9 system (a Type II system) has been found to be the best fit for experimental studies.

CRISPR-Cas9 consists of two key molecules that facilitate genome editing:

• The Cas9 enzyme: The Cas9 enzyme acts like a scissor cutting the genomic DNA at a particular location, thus, facilitating the addition or deletion of DNA base pairs.
• RNA molecules: Often called guide RNA (gRNA), this is a small piece of a pre-designed RNA sequence (about 20 nucleotide bases long) located within a longer RNA scaffold. It consists of CRISPR RNA or (crRNA) and tracrRNA. It guides the Cas9 enzyme to the specific target location where the cut is needed.

The working mechanism of the CRISPR-Cas9 system in labs is similar to that of bacteria. Its application in lab experiments involve the following steps:

• Select an organism for the experiment: Choose the organism whose genome you want to edit for your experiment. Note that CRISPR is not yet fully approved for practice on the human genome. It’s only allowed to be performed on animal models and isolated human cells.

If you are working on the treatment of genetic disorders, select animal models whose genomes are closest to humans.

• Select target gene location: Select the target gene location you wish to edit or alter. Collect more information on the target DNA sequence, such as its gene expression, GC content, phenotype, phenotypic variations, SNPs, and any other mutations. This will guide you in designing sgRNA and locating the PAM (protospacer adjacent motif) sequence.
• Select a CRISPR-Cas9 system: Select the suitable Cas9 nuclease and CRISPR sequences according to your experiment.
• Select and design the sgRNA: Computationally design sgRNA using the sequence information you extracted in the previous step.
• Synthesize and clone the sgRNA: Select a specific plasmid, insert the sgRNA in it, and clone it into multiple copies. Then, isolate the synthesized sgRNA copies from the plasmid.
• Deliver the sgRNA and Cas9: Insert the Cas9 and sgRNA into the target cell using gene transfer techniques such as the electroporation method.
• Validate the experiment: To learn if the manipulation is complete or the enzyme has cut at the target sequence, perform experiments like PCR, in vitro transcription, or next-generation sequencing.
• Culture the altered cells: Culture the modified cell lines using suitable culture media. When sufficient cell lines are obtained, insert them in the selected model organism.

In cells, the gap formed by the CRISPR-Cas system will be filled by either non-homologous end-joining (NHEJ) or by homology-directed DNA repair (HDR).

Image: A schematic diagram of DNA repair through NHEJ or HDR (using donor template) at the cleavage site.

• Gene expression study: Check the in vivo gene expression of the inserted DNA using RT-PCR or quantitative PCR.
• Analyze results: Analyze the results using computational techniques to determine success and study the phenotype change in the organism.

Common CRISPR Applications

CRISPR-Cas9 has several potential clinical applications:

• Gene therapy and treating medical conditions such as HIV, cancer, hepatitis B, hypercholesterolemia, and other diseases.
• Modifying stem cells that may then be re-injected into patients to repair damaged organs.
• In the food and agricultural industries to engineer probiotic cultures and protect industrial cultures (i.e., yogurt) from viruses.
• To engineer crops to improve their yield, drought tolerance, and nutritional properties.

It has several other applications in editing the genomes of somatic cells. However, its potential to edit the germline (reproductive cells) or human embryos is still a topic of debate among scientists because of its ethical implications.

CRISPR Limitations

CRISPR is one of the most dynamic gene-editing tools. Despite being more effective than other genome engineering techniques, the CRISPR-Cas9 system is not without its drawbacks. Some of its limitations are:

• It has a phenomenon of “off-target effects,” where DNA is cut at sites other than the intended target, resulting in unwanted mutations.
• Sometimes, even when the system cuts at the target, it might not cut at the precise location.
• It’s difficult to deliver the CRISPR-Cas9 system to a population of mature cells.
• It’s not 100% efficient; even when the cells take up the CRISPR-Cas9 system, it doesn’t always show any gene editing activity.

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CRISPR-Cas9 is a revolutionary genetic engineering tool that was adapted from prokaryotes and is used to alter DNA with precision and specificity for therapeutic use. It aids in manipulating an organism’s genome by cutting double-strand DNA at a particular location for the addition or deletion of desired sequences.

Today, CRISPR-Cas9 is used in biology, biotechnology, medicinal areas, therapeutic purposes, and food and agriculture.

However, performing these high-throughput experiments requires reagents and equipment of the best quality.

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