CRISPR-Cas9: A Targeted Genome Editing Technique
CRISPR is a family of DNA sequences which are present in the bacterium and these sequences contain parts of DNA from attacking virus. These viral DNA parts are used by the bacteria to control the future attacks and destruction of infected viral DNA. These specific sequence play important role in the defense system of the bacteria and provide basis for genome editing with permanent modification in genome.
CRISPR is denoted as Clustered Regularly Interspaced Short Palindromic Repeats. This CRISPR/Cas system is mainly present in prokaryotes and is part of their immune system to show resistance to the foreign genetic materials. These types of activities can be seen in phages and plasmids in the form of acquired immunity. The presence of these CRISPRs in the sequenced bacterial and archaeal genome is 40% and 90%, respectively.
The simplest version of this CRISPR/Cas system is CRISPR/Cas9. This system is comprised of nucleases like Cas9 and it is complexed with synthetic guided RNA also known as g-RNA inside the cell. This Cas9 nuclease with guided RNA cut the genome at desired location and lead to the removal of already existing gene and insertion of gene of interest in the genome.
Cas9 is an endonuclease and abbreviated as CRISPR associated protein 9. Cas9 is mainly used with synthetic guided RNA to produce a double stranded break within the strand of DNA at specific location. Cas9 is on one of the firstly discovered restriction nucleases also called molecular scissors.
How Does CRISPR/Cas9 Work?
Bacterial cells are mostly attacked by viruses and this CRISPR system primarily protects the bacterial cells by destroying the genome of the infecting viruses and provides care for ongoing and future infections. CRISPRs are regions present in the bacterial genome and help to protect the bacteria from invading viruses. These regions are mainly composed of short repeats of DNA with spacers. When a new unseen virus infects the bacterium, it will produce a new spacer among already existing spacers and serves as genetic memory. CRISPR sequence transcribes and produces short CRISPR RNA molecules. These molecules guide the bacterial machinery to identify the target sequences in the genome of invading virus and this machinery cuts and destroys viral genome (Fig 1).
Bacterial cells are mainly protected from viral attack through CRISPR immune system and there are three basic steps for this protection
- Genomic adaptation – by processing the DNA of attacking virus into small segments and add these CRISPR sequences in their own bacterial genome as new spacers.
- CRISPR RNA production – These spacers and repeats undergo transcription in bacterial genome and produce CRISPR RNA.
- Targeting the viral material – These CRISPR RNA molecules guide the bacterial machinery to destroy the material of invading virus because these RNA sequences are exact copies of viral DNA which are produced during adaptation process.
Applications of CRISPR/Cas System
Most important applications for those industries that use bacterial cultures for processing and this technique helps to make bacterial cultures more resistant to viral attack with efficient use. Manufactures mainly use different types of useful bacteria to produce different products like Streptococcus thermophilus is used to produce cheese and yogurt and mostly infected by viral attack. The original discovery of this technique mainly came from researchers, who were working on this organism in food production company. This technique can help to improve quality and quantity of food.
Genes have specific sequences and changes in the sequences will affect the cell biology. This technique is used extensively to make changes in the cellular genome of the organisms including humans, plants and animals. By using CRISPR, genes are being edited or deleted to make precise genetic changes in the genome of the target organism. In gene silencing, guided RNA is designed in such way that cuts both strands of target DNA at specific locations. Now, cell with broken DNA tries to repair but does it with errors and leads to an effective silence silencing. For gene editing, changes are made in repair template and it is incorporated into the target DNA during repair process (Fig 2).
Important medicinal application of CRISPR is to treat genetic diseases. The mutant genes can be corrected in animals with reversion of disease symptoms by using CRISPR. These mutant genes are replaced by normal genes (have correct sequences) to cure different heritable and infectious diseases in animal models.
Future of CRISPR
This technique has made rapid progress and CRISPR/Cas9 system has been extensively used as a tool for research in cellular and molecular biology due to its high efficiency, versatility and simplicity. The CRISPR nuclease systems are more precise and user friendly for genome editing and genomic engineering. Every technique takes some time in development, understanding and perfection. Although, much remains to be discovered but there is no doubt that CRISPR has now become a valuable technique in research and provides a future hope for the treatment of human diseases.
Dr. Zain Ul Abadeen
Ph.D. Researcher (Pathology), M.Phil, DVM, RVMP (Pak)
University of Agriculture, Faisalabad