Advanced: CRISPR-Cas9

Course Content

1. Basic laboratory practices

Basic safety, handling of micropipettes and safe disposal of laboratory wastes. To know more about how to handle micropipettes, watch this video or practice using this simulation.

2. Generation of double strand breaks using CRISPR Cas9 

A few base pair deletion in the plasmid gene of interest is achieved by sgRNA guided Cas 9 enzyme to introduce double stranded DNA breaks.

3. Purification of CRISPR Cas 9 treated DNA

The edited DNA is purified using a column-based kit and further used for setting up a ligation reaction.

4. Ligation

Plasmid subjected to CRISPR Cas9 induced double stranded DNA breaks is self ligated with the help of the enzyme DNA ligase. 

5. Transformation and phenotype analysis

Using the edited plasmid DNA, transformation is performed by heat shock and cells are plated onto antibiotic containing agar plates. Read the article on competent cells and transformation. 

6. Blue-white screening, clone analysis and confirmation

Colonies that grow after transformation are screened via blue-white technique and confirmed for the deletion of a few base pairs through plasmid DNA isolation and PCR. To understand the principle of blue-white screening, watch this video.

7. Isolation of plasmid DNA by alkaline lysis

Plasmid DNA extraction is a critical step in confirming whether the CRISPR Cas9 induced gene mutation was successful in the screened white colonies and there are no false positives. Read about alkaline lysis here. 

8. PCR-based confirmation of CRISPR Cas9 induced gene mutation

Using primers designed for the specific deleted region, Polymerase Chain Reaction (PCR) is done which will help in confirming the gene mutation. To know more about PCR, you can read this article.