Teaching Assistant

Skills
CRISPR-Cas9, PCR, cell culture, transfection, imaging, primer design, sequence analysis

Project Duration
3 months

1. Design of Guide RNA (gRNA): A single guide RNA molecule is designed to be complementary to a specific target DNA sequence within the genome. This gRNA guides the Cas9 enzyme to the target site.
2. Gene Synthesis: The gene sequence encoding the Cas9 protein is synthesized using molecular biology techniques, often based on bacterial systems. This synthetic gene is optimized for expression in the target organism.
3. Plasmid Construction: The synthetic Cas9 gene and the gRNA sequence are inserted into a plasmid, a small circular DNA molecule often used as a vector for introducing genetic material into cells.
4. Plasmid Amplification: The plasmid is replicated in bacteria through a process called plasmid amplification. This produces a large quantity of the plasmid DNA containing the Cas9 and gRNA sequences.
5. Purification of Plasmid DNA: The plasmid DNA is purified from the bacterial culture, ensuring that it’s free from contaminants.
6. Cell Culture Preparation: The target cells are cultured and prepared for transformation. This involves ensuring the cells are in a state where they can take up the foreign DNA.
7. Transfection or Electroporation: The purified plasmid containing Cas9 and gRNA is introduced into the target cells using techniques such as transfection (using chemical agents) or electroporation (using electrical pulses). This allows the cells to take up the CRISPR-Cas9 components.
8. Cas9-gRNA Complex Formation: Once inside the cell, the Cas9 protein and gRNA form a complex. The gRNA guides the Cas9 protein to the specific target DNA sequence.
9. DNA Cleavage and Repair: The Cas9 protein acts as molecular scissors and binds to the target DNA. It introduces double-stranded breaks (DSBs) at the desired location. The cell’s natural DNA repair mechanisms then come into play to repair the break. This can lead to gene knockout if the repair process introduces errors, or gene insertion if an external DNA template is provided.
10. Verification: The edited DNA is analyzed and verified using techniques such as polymerase chain reaction (PCR) and DNA sequencing. This confirms the successful modification of the target gene.
11. Functional Analysis: The effects of the gene modification on the organism or cells are studied to understand the functional consequences of the genetic alteration.