The field of Genetic Engineering specifically and Biotechnology has been the recent research topic of the Life Sciences in the current century. The advanced automated laboratories and successful sequencing of the complete Human Genome Project have led to the whole genome sequencing on a large scale resulting in cost-effective and timesaving cell-based phenotypic identifications. After the discovery of RNA interference, a gene accessing tool had remained the only genome screening technique until the early 2000s, as there was no alternative method developed till then. But with the constant emerging defects including off-target effects, variable intensities of efficient knockdown, etc. there was a need for a new, more advanced, and accurate technique for genome editing and screening. The more advanced genome editing techniques such as Zinc Finger Nucleases (ZNF)’s and Transcription Activator-like effector Nucleases (TALEN)’s back then were efficient but costly and time-consuming to engineer the process and took much time to process the whole genome on a large scale.
The CRISPR/ Cas-9 system has provided the solution. It proved to be efficient, cost-effective, and the most targeted technique discovered to date.
Introduction
Since the start of 2000, immense progress has been seen in Biomedicine both technically and scientifically. Among the certain “breakthrough” research discoveries of the year, CRISPR/Cas-9 has been prominent for its efficacy and precision back in 2015.
CRISPR stands for Clustered Regularly interspaced Short Palindromic Repeats while Cas-9 means CRISPR Associated System-9. Originally, CRISPR/Cas-9 is a responsive Immune system found in prokaryotes that possesses resistance against any foreign genetic material to say it could be viral or any other plasmid vector, providing a form of gained immunity to the organism. At the very start of its discovery, CRISPR/Cas-9 was a lesson to learn in segments of bacterial DNA having short, insistent base sequences. Later, in 2012, a tool was presented to cut DNA by Jennifer and her team at the University of California, Berkeley. Further, the group of Feng Zhang at Board Institute in Boston claimed to use this technique for eukaryotic genome editing in-vitro on a mouse or human cells in 2013.
Working of CRISPR/Cas-9
Precisely, CRISPR/Cas-9 is a specialized DNA sequence containing two distinct features;
a) Nucleotide repeat sequence
b) Spacer
The Nucleotides are building blocks of DNA, while Spacers are those tiny bits of DNA that are present among Nucleotide sequences in the CRISPR region. The concept of CRISPR/Cas-9 has arisen as a bacterial defense mechanism against viral attack. Some fragments of the viral genome are to remain safe by bacteria as a “memory” in the CRISPR Spacer region so that on re-occurrence of the same viral attack, it can be a deal more quickly and efficiently as Cas-9 protein cuts down the viral genome as soon as it attacks. Guide RNA (gRNA) guides the Cas-9 protein to cut the sequences at the targeted position.
Why CRISPR Became the Hottest Ongoing Research Topic
At the start, Scientists were curious about what exactly is the mechanism of CRISPR/Cas-9? Until 2012, when Jennifer Duodena of the University of California, Berkeley found out by performing certain experiments along with Emmanuelle Charpentier of Umeå University in Sweden. They provided the bacterial cell with “artificial RNA” whose sequence was almost complementary to the viral genome that had attacked bacteria earlier, and noted that Cas-9 protein recognized the sequence and chopped it down into fragments. They found out that this mechanism can be useful in cutting, silencing, editing, adding, or removing any desired gene at any desired place within the genome.
This discovery revolutionized the whole genetic engineering field as gene editing has never been this much precise and accurate until that date. With the further advancements made, Feng Zhang from Board Institute, Boston, and Harvard’s George Church published articles in Science showing how to use the powerful CRISPR/Cas-9 for human genome editing and other eukaryotic genomes, including mice.
With the help of this amazing discovery, one can remove defective genes, or replace them precisely and accurately. We can add new and healthy genes at the desired place, for instance, a scientist can easily replace the defective or non-functioning genes causing Huntington’s disease with ‘good’ genes.
The prime reason for CRISPR/Cas-9 to be the revolutionary discovery, as they prove it to be most precise and accurate. Cas-9 exactly where you want it to target genes. This technique is unbelievably cost-effective and inexpensive.
CRISPR/Cas-9 has been the hottest ongoing research topic as there were only a few dozens of research articles published till 2011, but at the end of 2018, there were over 17,000 published research papers that are still counting. As J. Duodena said that it has been such a fast-moving field, even she finds it difficult to keep up.
Where can one use CRISPR?
Basically, the map for the human genome was accomplished back in 2003 but scientists still did not know exactly which gene expresses certain functions but with the help of CRISPR/Cas-9, one can easily identify by knocking out a specific gene and then observing which trait has been pretentious. It speeds up the genome screening process. A 2016 article in Nature Biotechnology by Rodolphe Barrangou and Duodena enlisted about of possible future applications:
- Crop gene editing for more nutritious food
- Develop the latest tools to replace defective genes
- Novel antibiotics and antiviral production
- Develop Gene Drive to alter whole specie’
- Insert genes to remove the harmful species entirely
- Restrains to the CRISPR/Cas-9 Technique
Although it has been one of the hottest research topics of the recent century, it has certain limitations to it. It is cost-effective and cheap compared to the previous techniques used, yet sometimes it can go off target causing DNA cuts at the sites other than the targeted ones. As Church said at Live Science, the chief drawback of CRISPR/Cas-9 is that it is not 100 percent proficient. Editing in genes may diverge, sometimes up to 50-80%; they may show varying intensity in the gene editing.
Bottom line
CRISPR/Cas-9 is one of the very recent innovations in genetic engineering. It has shown potential applications and benefits for genome editing that may provide the cure for genetic disorders and can help create better crop varieties with enriched nutrients and crop yield in the coming days. It can also help to introduce the required features in the developing embryo.
