Day to day life is being changed for centuries with innovation. Science took the human to a new direction by every century given a tremendous technology and method of life sustainability. There are thousands of scientists with associates working in the labs round the clock and coming up with innovations. Some of them give a new direction of scientific innovation, so I am writing about one of them today, and the latest is CRISPR CAS 9 Technology. The basics of CRISPR CAS 9 (Clustered regularly interspaced short palindromic repeats – CRISPR associated protein 9).
This phenomenal work is giving a different direction to the Gene Editing system. Let us see how this works
Discovery of CRISPR
Although CRISPR/cas9 emerged as a powerful molecular scissor for genome editing in 2012 when George Church, Jennifer Doudna, Emmanuelle Charpentier, and Feng Zhang utilized it to modify targeted regions of genomes, it was first found in E.coli way ahead in 1987 by a Japanese scientist, Yoshizumi Ishino, and his team. Henceforth, it was observed in various bacteria such as M. tuberculosis, Streptococcus thermophilus, Etc., and archaeal genomes, whereas Researchers Francisco Mojica and Ruud Jansen referred to the unusual series of repeated sequences interspersed with spacer sequences as CRISPR.
Adaptive Immunity in Bacteria
Later in the early 2000s, Mojica and coworkers revealed that bacteria develop an adaptive immunity against bacteriophage and viruses by harboring homologous spacer sequences in the CRISPR region. Briefly, when viruses inject their DNA into the bacterial cell, cells acquire a small piece of foreign DNA (spacer) integrates into an interspaced palindromic region known as the CRISPR locus. The bacteria transcribe this sequence to generate CRISPR RNA (crRNA), which combines with another type of RNA called Tracer RNA (trRNA) and inactive CRISPR-associated Protein (Cas), thereby activating the Cas protein. Moreover, as this active CRISPR/Cas surveillance complex encounters the foreign DNA (virus) having complementary sites to crRNA, CRISPR/Cas can cut the DNA leading to the destruction of foreign genomic material. Thus securing immunity against the viral/bacteriophage.
FIGURE 1: Natural vs. Engineered CRISPR systems. (Design Credit: https://sites.tufts.edu)
CRISPR/Cas9 as Gene-editing tool
In the early 21st century, CRISPR/Cas9, a standardized gene-editing tool, has overwhelmed researchers with an assurance of making gene editing simpler, faster, cheaper, and more accurate & efficient than other genome editing methods. Jennifer Doudna and Emmanuelle Charpentier got a Nobel award for discovering one of gene technology’s sharpest tools: the CRISPR/Cas9 Molecular Scissor in 2020. With the publication of Charpentier discovery, there was a bloom in Gene modification research, enabling the scientist to correct, delete (Knock out), Add/replace (Knock in) in various sectors such as Public health, Agriculture, and Biomedicine.
Researchers program a CRISPR array able to be transcribed into small guide RNA (gRNA) and tracerRNA (trRNA)having an anti-repeat sequence that allows it to base-pair with the repeats of gRNA. In the vicinity of there are CRISPR-associated genes that encode the Cas protein, which stabilize the duplex of gRNA and trRNA. Even though the gRNA with targeting information directs Cas9, Proto Spacer Adjacent motif (PAM seq.) plays a vital role for Cas9 to recognize and cleave DNA. Cas9 can bind and release DNA quickly, thus searching through a vast stretch of DNA.
Researchers program a CRISPR array able to be transcribed into small guide RNA (gRNA) and tracerRNA (trRNA)having an anti-repeat sequence that allows it to base-pair with the repeats of gRNA. In the vicinity of there are CRISPR-associated genes that encode the Cas protein, which stabilize the duplex of gRNA and trRNA. Even though the gRNA directs Cas9 with target information, the Proto Spacer Adjacent motif (PAM seq.) plays a vital role for Cas9 to recognize and to cleave DNA. Cas9 can bind and release DNA quickly, thus searching through a vast stretch of DNA. As soon as it finds the PAM site (NGG, CCN on complementary strand) and complementarity for binding gRNA, the strands of DNA melt apart, driving RNA DNA helix formation inside the Cas9 protein. If the helix is perfect or close to perfect, the nuclease is triggered to cut dsDNA, generating a blunt double-stranded break (DSB). At this point, DSB is repaired either by Non Homologous End Joining (NHEJ) or homologous repair (HR), harnessed to achieve precise gene modifications or gene insertion. In Eukaryotic cells, this mechanism is equally effective at triggering double-stranded breaks that can be repaired and trigger changes in the format of genome editing.
Design credit: Arora and Narula
Application
According to recent research (2021), Lead author Sharon Lewin from Australia’s Peter Doherty Institute for Infection and Immunity has successfully used CRISPR gene-editing technology to prevent the transmission of the Sars-CoV-2 virus. They have used the CRISPR-Cas13b tool to destruct the COVID-19 virus upon recognizing the virus and thus activation. It also has the potential to solve the world’s most dire problem of human health and food security.
Reference
Arora, L., & Narula, A. (2017). Gene editing and crop improvement using CRISPR-Cas9 system. Frontiers in plant science, 8, 1932.
Komor AC, Badran AH, Liu DR. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell. 2017 Apr 20;169(3):559. doi:10.1016/j.cell.2017.04.005. PubMed: 28431253.