CRISPR Cas9 : How CRISPR can be performed in the lab ?

Introduction

This tutorial provides a comprehensive step-by-step guide on performing CRISPR-Cas9 in the laboratory, based on the video by Animated Biology with Arpan. CRISPR-Cas9 is a revolutionary gene-editing technology that allows for precise modifications to DNA, making it a powerful tool in genetic research, medicine, and biotechnology.

Step 1: Design the Guide RNA

• Identify the target gene you wish to edit.
• Design a guide RNA (gRNA) that matches a specific sequence in the target gene.
• Ensure the gRNA is approximately 20 nucleotides long and includes a protospacer adjacent motif (PAM) sequence (typically "NGG").
• Use online tools and databases to assist in gRNA design.

Step 2: Construct the CRISPR-Cas9 Plasmid

• Obtain the Cas9 nuclease and the designed gRNA sequence.
• Use molecular cloning techniques to insert the gRNA sequence into a plasmid that expresses both the gRNA and Cas9.
• Confirm the successful construction of the plasmid through restriction enzyme digestion and sequencing.

Step 3: Transfect the Target Cells

• Choose an appropriate method for transfecting the plasmid into your target cells (e.g., lipofection, electroporation, or viral delivery).
• Prepare your cells in culture, ensuring they are in the logarithmic growth phase for optimal transfection efficiency.
• Follow the protocols for your chosen transfection method carefully to maximize uptake.

Step 4: Selection and Screening of Edited Cells

• After transfection, allow time for the cells to express the Cas9 and gRNA.
• Apply selection markers (if available) to identify successfully edited cells.
• Use techniques such as PCR and Sanger sequencing to screen for the presence of desired mutations in the target gene.

Step 5: Validate the Gene Editing

• Validate the intended edit by performing additional sequencing on the target region.
• Assess the efficiency of the edit and check for off-target effects using methods such as T7 endonuclease I assay or whole-genome sequencing.
• Analyze the phenotypic changes resulting from the gene edit to confirm functional impact.

Conclusion

In this tutorial, we covered the essential steps for performing CRISPR-Cas9 in the laboratory, from designing the gRNA to validating gene edits. Understanding these steps is crucial for researchers looking to harness this powerful gene-editing technology. As a next step, consider exploring applications of CRISPR-Cas9 in your area of study, or review the latest advancements in CRISPR technology to stay updated on best practices and innovations.