Nobel Prize in Chemistry 2020
Nobel Prize in Chemistry 2020
Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry 2020 for discovering one of gene technology’s sharpest tools i.e. the CRISPR/Cas9 genetic scissors.
- Emmanuelle Charpentier was studying a bacteria called Streptococcus pyogenes and noticed a previously unknown molecule called tracrRNA.
- The tracrRNA was part of the bacteria’s immune system and it helps the bacteria destroy viral DNA.
- In 2011, Charpentier and Doudna succeeded in recreating the bacteria’s scissors and reprogramming it and then proved that they can now use these scissors to cut any DNA molecule at a required site.
CRISPR/Cas9 genetic scissors:
- CRISPR is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats.
- The CRISPR-Cas9 system consists of two molecules that make an edit in the DNA.
- Cas9: An enzyme that functions as a pair of ‘molecular scissors’. It has the ability to cut the two strands (sense and anti-sense) of DNA at a specific location in the genome.
- guide RNA (gRNA): A short (20 bases or so) piece of pre-designed RNA sequence located within a longer RNA scaffold. The gRNA guides the Cas9 to the planned part of the genome ensuring the right place is cut.
- The gRNA has bases that are complementary to the target sequence in the DNA like a very specific lego piece. This in theory ensures that the gRNA binds only to the desired sequence and not somewhere off-target.
- The Cas9 scissor enzyme is guided to the desired location and snips across both strands of the DNA. Once this happens the cellular mechanism understands that the DNA is damaged so initiates a repair mechanism. At this juncture, it is possible to use this opportunity to introduce changes to the genes of their design.
Possible Applications with CRISPR-Cas Systems:
- With the use of a target-specific CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), or a fused format called a single guide RNA (sgRNA), locations within complex mammalian genomes can be targeted by the Cas9 endonuclease for a double-stranded break.
- The crRNA, tracrRNA, and sgRNAs can either be transcribed intracellularly, in vitro transcribed or custom synthesized and introduced through transfection.
- The Intracellular expression of Cas9 endonuclease can be accomplished by plasmid or integrated lentiviral expression vectors driven by constitutive or inducible promoters.
DNA-free CRISPR-Cas9 gene editing:
- The system uses no CRISPR-Cas9 components in the form of DNA vectors i.e. each component is either RNA or protein.
- The use of DNA-based Cas9 or guide RNA expression systems carries with it the possibility of undesirable genetic alterations due to plasmid DNA integration at the cut site or random lentiviral vector integrations.
- A DNA-free gene editing system can be a good choice for creating engineered cell lines.
Homology-directed repair (HDR):
- The CRISPR-Cas9 induced double-strand break can also be used as an opportunity to create a-knockin, rather than a target gene knockout.
- The precise insertion of a donor template can alter the coding region of a gene to “fix” a mutation, introduce a protein tag, or create a new restriction site.
Embryonic stem cell and transgenic animals:
- CRISPR-Cas systems can be used to rapidly and efficiently engineer one or multiple genetic changes to murine embryonic stem cells for the generation of genetically modified mice.
Transient activation of endogenous genes (CRISPRa or CRISPR on):
- By employing a Cas9 mutant that cannot cut DNA and to which a transcriptional activation domain has been fused, the expression of endogenous genes can be up-regulated by targeting the Cas9 fusion protein to the promoter region of an endogenous target gene, or multiple genes simultaneously.
Source: Indian Express