CRISPR-Cas9: Unlocking the Genome

The discovery of CRISPR-Cas9 opened doors to explore the area of genomics in a new way. It shows the numerous possibilities to unlock the genome and lock it again through it’s highly specific and accurate nature. It’s major application in medical field as a therapeutic option for rare genetic disorders fascinated me a lot and thus, it became my area of research interest. Let’s deep dive into the fascinating world of CRISPR-Cas9 and explore the possibilities it holds.

• Discovery

  CRISPR-Cas9 was discovered as an adaptive immunity in bacteria against bacterial viruses or bacteriophages. When a bacteriophage infects a bacteria, it’s genetic material gets inserted in the bacterial genome becoming a part of it. During the subsequent exposure with the same bacteriophage, bacteria can tolerate it as it retains the memory inside its genome.

• Mechanism of action

  CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats are the elements or loci in the genome of bacteria, plants and animals. Their nature can already be understood by its full name. They are short, clustered, palindromes spaced with repeats.

  Cas9 refers to CRISPR-associated genes that encodes the nuclease enzyme, specific towards and targets the CRISPR locus. Cas9 is more extensively studied Cas enzyme isolated from the bacterium Streptococcus pyogens due to its simplicity, abbreviated as SpCas9.

  As discussed in discovery, when a bacteriophage infects a bacteria, it’s genome gets inserted into the bacterial genome — CRISPR locus which further transcription produces crRNA (CRISPR-derived RNA). This crRNA along with the Cas9 enzyme targets the same sequence it transcribed from, a process known as RNA interference.

  Cas9 being highly specific nuclease recognizes it’s target and makes a cut or incision in the DNA. For accurate recognition, there is a PAM sequence present in the CRISPR locus. As CRISPR locus consists of a leader sequence, spacers and repeats, the spacer that are destined to become a part of bacterial genome from a bacteriophage is called a Protospacer. PAM stands for Protospacer Adjacent Motif which lies just next to the protospacers. So, the PAM sequence for Cas9 is 5’-NGG-3’ where ‘N’ can be any nucleotide from A, T, G and C.

  Cas9 has two nuclease domains — HNH domain and RuvC domain. HNH domain for the cleavage of complementary strand and RuvC for the cleavage of non-complementary strand. Thus, it introduces double strand breaks (DSBs) in DNA after the cleavage. But not alone, it requires crRNA and a another RNA component i.e. transactivating crRNA (tracrRNA) transcribed from the different locus for the accurate and specific action.

  Lastly, the DSBs introduced by Cas9 are repaired by the cells endogenous DNA repair pathways.

  

• Applications

  CRISPR-Cas9 has wide applications for genome editing in bacteria, plants and animals. To treat antibiotic resistance, to develop high yield crops, to model diseases in-vitro and to enhance desired traits or characters are the major focus areas. But the area that is trending and fascinated me is to treat or cure rare genetic disorders which were incurable before. Being someone with a rare neuromuscular genetic disorder — Muscular Dystrophy, this became my area of interest. As we understood the mechanism of CRISPR-Cas9 technology, let’s now have a look at its broad application in therapeutics.

  First step is the development of single guide RNA (sgRNA) that is complementary to the target genetic sequence to be corrected. It’s important because Cas9 requires RNA component to bind accurately to its target. Cas9 alone is a nuclease, it will cleave DNA anywhere in the genome if sgRNA is not used. sgRNA is nothing but crRNA and tracrRNA joined by linker loop. Thus, Cas9 with sgRNA will recognize it’s target and produce DSBs in the DNA. These DSBs are repaired by two pathways — Non Homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Both of these pathways have their own advantages and disadvantages.

  NHEJ is error prone repair pathway producing insertions and deletions (indels) in the gene and thus, it disrupts the target gene known as gene knockout. So, it can be said that NHEJ pathway can be used for production of gene knockouts in mouse models or human cells. In terms of rare genetic disorders, NHEJ can be used to knockout the pathogenic (disease causing) dominant gene.

  On the other hand, HDR uses the more accurate Homologous Recombination pathway. This pathway can correct a gene mutation by inserting a corrected sequence known as knock-in or gene insertion at a target gene locus. Here, donor template which contains the correct sequence along with homologous sequence is incorporated with sgRNA and Cas9. HDR pathway can be used in recessive disorders where normal gene is absent. So, this pathway is for the gene correction.

  Despite the specificity and accuracy, there are “off-targets” seen. This is the major disadvantage of this technology. CRISPR components can bind the other sites rather than the target sites resulting in knockout of the healthy gene or inserting a gene sequence at a undesired location known as off-target effects. These can be lethal too like inducing oncogenic mutation giving rise to cancer.

  

• Future Advancements

  CRISPR-Cas9 has a vast scope in the field of precision genome editing. The studies are going on to enhance the accuracy and efficacy of the technology majorly to reduce the off-targets and recognize the target with the greater precision. There is ongoing search for alternative Cas variants from microorganisms other than Cas9 for accurate recognition of target. Also, creating different PAM sequence by mutation and checking for the recognition.

  Despite of these, Cas9 holds a great promise because of its specificity. A variant of Cas9 is designed by inducing mutation in it known as dead Cas9 (dCas9) in which there is no nuclease activity present. Thus, dCas9 cannot cleave the DNA but recognize the target sequence precisely. So, it’s used for the epigenetic modifications like switch “on” or “off” the desired gene. Likewise, dCas9 can be combined with activation domain, repression domain, epigenetic modifier, base editor and fluorescent protein to make the required changes, corrections or identifying location of specific sequence in the genome of cells according to our necessity. Another, Cas9 variant known as “nickase” in which only one nuclease domain (HNH) is active and the other domain (RuvC) is inactivated by mutation. This increases the probability of HDR repair pathway eliminating the mutagenic NHEJ repair pathway.

  Future is bright for CRISPR-Cas9 and its broad application in the field of genomics. I think that future is full of exploring CRISPR-Cas9 technology in bacteria, plants and animals at an affordable cost and less time with the benefits that are still beyond the imagination.

CRISPR-Cas9 is the world of fascination that makes genomics more accessible and trending subject of biology with the numerous possibilities to be uncovered. Extensive research is being carried out in this area from lab bench to bedside translating it on the grassroots. CRISPR-Cas9 has increased the scope of other areas too like precision medicine or translational medicine that has to be kept eye on. Specifically, rare genetic disorders that were considered to be incurable but the invent of CRISPR-Cas9 opened the door as a ray of hope to cure or treat such disorders in the near future. Current studies have already proved its potential on a global scale. Though, less cases exists now but research is the answer for the scalability and efficiency of this technology.

I’ve tried my best to cover most of the CRISPR-Cas9 technology and it’s applications in the real world but still it’s a review according to me. I think that I’ve defended my area of interest quite well by my knowledge. I hope that you guys like this blog and find it helpful in your research journey. Thank you so much for taking time and reading it…