BIOL 301: Virtual Genetics Lab III (Gene Editing & Analysis)
Welcome to the third installment of the BIOL 301 Virtual Genetics Lab series. In this lab, you will transition from classical inheritance patterns to modern molecular genetics. You will utilize industry-standard bioinformatics software and virtual simulation tools to design, execute, and analyze a gene-editing experiment using CRISPR-Cas9 technology. Lab Objectives By the end of this lab module, you will be able to:
Design highly specific single-guide RNAs (sgRNAs) targeting a gene of interest.
Simulate the delivery and mechanism of the CRISPR-Cas9 system in a host genome.
Analyze gene knockout efficiency using sequencing data chromatograms.
Evaluate potential off-target mutations using computational alignment tools. Part 1: Computational Target Selection & sgRNA Design
Modern gene editing relies on precision. Your first task is to navigate the online genome browser to locate your assigned target gene: Human HBB (Hemoglobin Subunit Beta). Step 1: Locating the PAM Site
CRISPR-Cas9 requires a Protospacer Adjacent Motif (PAM) sequence to bind to target DNA. The SpCas9 enzyme specifically recognizes the 5’-NGG-3’ motif. Open the provided benchling or NCBI locus link. Locate Exon 1 of the HBB gene.
Scan both the sense and antisense strands for valid PAM sites. Step 2: Evaluating Guide RNA Efficiency
Once you identify three potential 20-nucleotide target sequences directly upstream of a PAM site, you must evaluate them based on two critical scores:
On-Target Score: Predicts the cutting efficiency of the Cas9 enzyme at your chosen site.
Off-Target Score: Measures the probability that your sgRNA will accidentally bind and cut elsewhere in the genome.
Select the guide RNA that optimizes high on-target efficiency while minimizing off-target risks. Part 2: The Virtual Molecular Bench
After finalizing your sgRNA design, you will transition to the virtual lab bench simulator to prepare your molecular vectors.
[sgRNA Insert] + [pSpCas9 Plasmid Vector] │ ▼ (Restriction Digestion & Ligation) [Recombinant Expression Vector] │ ▼ (Virtual Electroporation) [Target Cell Culture Host] Protocol Simulation Steps
Digestion: Digest the plasmid backbone using specific restriction enzymes to create sticky ends.
Ligation: Subclone your synthesized sgRNA oligos into the prepared plasmid vector using T4 DNA Ligase.
Transformation: Introduce the recombinant plasmid into E. coli cells for amplification via virtual heat-shock.
Transfection: Harvest the purified plasmids and introduce them into your target human cell culture via electroporation. Part 3: Mutation Analysis & Sequencing
After allowing 48 hours for cell incubation and genome editing to occur, you will harvest the cellular DNA to verify your results. Sanger Sequencing Analysis
You are provided with a digital Sanger sequencing file (.ab1) derived from your edited cell line. Upload the file to the virtual sequence alignment tool.
Align your edited sequence against the wild-type HBB reference gene.
Identify the Mutation: Look for overlapping, chaotic peaks immediately 3 to 4 base pairs upstream of your PAM site. This indicates a mixed population of cells with various insertions or deletions (indels) caused by Non-Homologous End Joining (NHEJ). Post-Lab Assignment & Reporting
Your lab report must be submitted as a PDF via the course portal by the start of next week’s session. Ensure your report contains the following elements:
Introduction: A brief overview explaining how the Cas9 endonuclease introduces double-strand breaks (DSBs) and how NHEJ leads to gene knockouts.
Results Table: Include your selected 20-bp sgRNA sequence, its corresponding PAM sequence, and its predicted on-target and off-target scores.
Chromatogram Screenshot: Highlight the exact site of the double-strand break on your sequencing trace file.
Discussion: Explain whether your virtual edit successfully disrupted the reading frame of the HBB gene (e.g., did it cause a frameshift mutation?). Discuss the real-world therapeutic implications of editing this specific gene.
If you run into technical glitches with the plasmid map editor or the sequencing alignment tool, please post your questions in the course discussion forum or visit your TA during virtual office hours.
To help you get started on your post-lab report, please let me know:
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