Programmable DNA cleavage using CRISPR–Cas9 enables us efficient, site-specific genome engineering in single cells and whole organisms. Versatile CRISPR-enabled genome editing has been used in many ways, such as controlling transcription, modifying epigenomes, conducting genome-wide screens and imaging chromosomes.
CRISPR systems are already being used to reduce genetic disorders in animals and are likely to be used soon in the clinic to treat human diseases of the eye and blood. Two clinical trials using CRISPR-Cas9 for targeted cancer therapies have been approved in China and the United States. Beyond biomedical applications, these tools are now being used to expedite crop and livestock breeding, engineer new antimicrobials and control disease-carrying insects with gene drives.
- The CRISPR Cas9 technique is one of a number of gene-editing tools. Many favor the CRISPR Cas9 technique because of;
- Its high degree of flexibility and accuracy in cutting and pasting DNA
- It makes it possible to carry out genetic engineering on an unprecedented scale at a very low cost
- It is used to manipulate many different genes in a cell line, plant or animal very quickly, reducing the process from taking a number of years to a matter of weeks
- DNA repair mechanisms
The technique is already being explored for a wide number of applications in fields ranging from agriculture through to human health. In agriculture it could help in the design of new grains, roots and fruits.
The importance of the CRISPR/Cas9 was recognized with the awarding of the Nobel Prize in Chemistry to Jennifer Doudna and Emmanuel Charpentier on 7th October 2020.
The CRISPR-Cas9 system works similarly in the lab. Researchers generate a small piece of RNA with a short “guide” sequence that binds to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location.
The protein typically binds to two RNA molecules crRNA and another called tracrRNA (or “trans-activating crRNA”). The two then guide Cas9 to the target site where it will make its cut. This expanse of DNA is complementary to a 20-nucleotide stretch of the crRNA.
Once the DNA is cut, the cell’s natural repair mechanisms kick in and work to introduce mutations or other changes to the genome. There are two ways this can happen. According to the Huntington’s Outreach Project at Stanford (University), one repair method involves gluing the two cuts back together. This method, known as “non-homologous end joining,” tends to introduce errors. Nucleotides are accidentally inserted or deleted, resulting in mutations, which could disrupt a gene. In the second method, the break is fixed by filling in the gap with a sequence of nucleotides. In order to do so, the cell uses a short strand of DNA as a template. Scientists can supply the DNA template of their choosing, thereby writing-in any gene they want, or correcting a mutation. The CRISPR-Cas9 system consists of two key molecules that introduce a change into the DNA. These are:
An enzyme called Cas9. This acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed.
A piece of RNA called guide RNA (gRNA). This consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome. The desired piece of DNA that is inserted after the break.
The Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA.
At this stage the cell recognizes that the DNA is damaged and tries to repair it. Scientists can use the DNA repair machinery to introduce changes to one or more genes in the genome of a cell of interest.