The development of CRISPR has unlocked an innovative way to improve our livelihood by giving us a solution to massive issues that once plagued our greatest minds. This is especially true with the application of CRISPR in the agricultural sector, specifically in food security, since gene editing can improve yield by simply enhancing the crop’s traits and reducing its susceptibility to diseases. Meanwhile, in research, the application of CRISPR allows researchers to investigate the roles of individual genes to correct the mutations present in genetic disorders, which up until now have had no permanent cure. CRISPRs have been used in so many sectors and are currently being integrated into so many more. As this gene-editing phenomenon continues to aid our civilization in curing the problems that we once thought to be impossible, the more CRISPR will develop, one day allowing us to change essentially what makes us, human; our genomes.
What is CRISPR?
CRISPR, which is short for clustered regularly interspaced short palindromic repeats, is a technology that is used to selectively modify the DNA of living organisms. It was developed by adapting a naturally occurring editing system found in the DNA sequences of prokaryotic organisms. Using CRISPR, scientists can quickly create models of animal cells, which can be used to research diseases at a faster rate. It is also recently being developed in hopes of being used as a rapid diagnostic tool.
The Discovery of CRISPR
In 1987, unusual repetitive DNA sequences were discovered by Yoshizumi Ishino. These sequence patterns were then reported in many other prokaryotic organisms, which were later identified as CRISPR. Later, the connection between CRISPR and Cas proteins was discovered, which resulted in joint efforts in comparative genomics, structural biology, and advanced biochemistry to understand and develop CRISPR. The evolution of CRISPR took multiple decades, the current CRISPR-Cas 9 was co-invented in 2011 by Jennifer Doudna and Emmanuelle Charpentier, and they received the 2012 Nobel Chemistry Prize for their discovery.
Components of CRISPR
CRISPR is made up of many components such as:
CRISPR array: is a feature of the CRISPR systems found in the genome of prokaryotes, consisting of short, repeated DNA sequences that are separated by spacer sequences. The spacer sequences are derived from fragments of plasmid DNA that the organism has incorporated into its genome, forming a type of acquired immunity.
Cas proteins: are a key component of the CRISPR system, playing various roles in the recognition, processing, and targeting of foreign DNA.
Guide RNA (gRNA): is a synthetic RNA molecule that directs the Cas nuclease to the DNA sequence targeted for editing.
Protospacer Adjacent Motifs (PAM): are specific DNA sequences next to the target DNA that are necessary for Cas proteins to recognize and bind to the target DNA.
DNA Repair Machinery: the Cas proteins cause the double-strand break in the target sequence; the repair machinery is activated in this instance.
Mechanisms
Adaption: The proteins Cas 1 & 2 are involved in the adaptation of CRISPR, forming a complex that recognizes and divides the foreign DNA, capturing spacers (short segments), and adding them to the CRISPR array.
Transcription and Processing: the CRISPR array is transcribed by RNA molecules, that are processed into individual crRNAs, and then a trans-activity CRISPR RNA (tracrRNA) hybridizes with the crRNA, forming the duplex RNA structure that guides the Cas9 nuclease.
Interference: the crRNA and tracrRNA bind to the Cas protein forming an RNP complex that scans the bacterial genome for sequences complementary to the crRNA spacer. Then the Cas protein uses the cRNA spacer to pair with the complementary sequences in the target DNA. When the crRNA-Cas complex recognizes the target DNA sequences and binds, the Cas nuclease induces the double-strand break, triggering the DNA repair mechanisms.
DNA Repair: The primary repair path is triggered which just directly links the broken DNA back together. This can result in small insertions or deletions from the target site, disrupting the functioning of the target gene.
The problems faced
One of the major concerns regarding the CRISPR-Cas system is the effects caused when it is off target, in which the CAs protein may split the DNA sequences that are similar to the target sequences, leading to unintended mutations in other parts of the genome. This could cause a lot of harmful and undesirable effects on the genome. To combat this concern, researchers are developing ways and strategies to improve the specificity of CRISPR-Cas.
Another issue is that the delivery to target cells is a massive hurdle because if CRISPR is to be used therapeutically in humans, then specific delivery methods have to be developed to ensure CRISPR components can reach the genomic targets inside different types of human cells. This is an essential issue because there is a large risk of triggering the immune response, leading to the clearance of the CRISPR which affects its efficiency. However, it should be noted that the efficiency and specificity of CRISPR-CAS are very dependent on the cell type and chromatin structure of the target DNA.
Written by: Trisha Saju
コメント