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.
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:
Image: An illustration of the working mechanism of the CRISPR-Cas system in bacteria against bacteriophages.
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 working mechanism of the CRISPR-Cas9 system in labs is similar to that of bacteria. Its application in lab experiments involve the following steps:
If you are working on the treatment of genetic disorders, select animal models whose genomes are closest to humans.
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.
CRISPR-Cas9 has several potential clinical applications:
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 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:
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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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