Lecture Notes: CRISPR-Cas and Knockout Lab Overview

Introduction

• Presenter: Alison from Mini PCR Bio, curriculum team
• Focus: New CRISPR-Cas hands-on lab for students to modify DNA in living cells
• Goal: Provide students with practical genome editing experience

Background on Genome Editing and CRISPR-Cas

• Genome Editing: Changing DNA sequence in cells
  • Methods include adding/removing sequences, swapping bases
• CRISPR-Cas System: Developed between 2012-2013, revolutionized genome editing
  • Advantages: Simplicity, adaptability, specificity
  • Recognition: 2020 Nobel Prize in Chemistry
• Natural Origin: CRISPR-Cas exists in bacteria and archaea as an antiviral defense

Components of the CRISPR-Cas System

• Cas Protein: Enzymes like Cas9 cut DNA
  • Guided by small RNA to specific DNA sequences
• Guide RNA: Determines where Cas9 cuts, binds to DNA
  • Approximately 20 base sequence matches target DNA
• Process:
  • Guide RNA binds Cas9, DNA is unzipped
  • Cas9 cuts if guide RNA matches DNA sequence

Importance of Specificity and Function

• Specificity: Essential to target precise DNA sequences
  • 20 base sequence specificity reduces off-target effects
• Knocking Out Genes: Understanding gene functions by disabling them
  • CRISPR-Cas allows gene knockout in any organism

Example: Snail Gene Knockout

• Study: Used CRISPR-Cas to knockout LSDIA1 gene, affecting shell spiral direction
• Outcome: Demonstrated gene's role in shell development
• Broader Implications: Insights into left-right asymmetry could have human clinical applications

Mini PCR Knockout Lab Overview

• Objective: Knockout a gene in bacteria using CRISPR-Cas
  • Target: LacZ gene associated with color phenotype
• Experiment Steps:
  1. Introduce Cas9 and guide RNA into bacteria via plasmids
  2. Use transformation involving calcium chloride and heat shock
  3. Select transformed bacteria using antibiotic resistance
  4. Observe results via blue-white screening
• Results:
  • Blue colonies indicate functional LacZ gene
  • White colonies indicate successful knockout

Educational Resources and Implementation

• Preparation: Simple, complete in 3 days
  • Day 1: Teacher prep, Day 2: Student transformation, Day 3: Results observation
• Teaching Aids: DNA Dots, paper activities for better understanding
• Additional Learning: PCR genotyping add-on to confirm gene knockout

Concluding Remarks

• Resource Availability: Free resources and educational materials available on Mini PCR website
• Future Availability: Knockout lab expected early 2021
• Contact: Updates through QR code or links

These notes summarize the main points and workflow of the CRISPR-Cas system and its application in educational lab settings, providing a comprehensive overview of the knockout lab designed by Mini PCR Bio.