CRISPR/Cas9: A Genome Editing Tool

A genome editing tool derived from bacteria. Consists of a guide RNA (gRNA) 1, which is complementary to a target DNA sequence, and an endonuclease (Cas9), which makes a single- or double-strand break at the target site 2. Imperfectly cut segments are repaired by nonhomologous end joining (NHEJ) → accidental frameshift mutations (“knock-out”) 3A, or a donor DNA sequence can be added to fill in the gap using homology-directed repair (HDR) 3B. Potential applications include removing virulence factors from pathogens, replacing disease-causing alleles of genes with healthy variants (in clinical trials for sickle cell disease), and specifically targeting tumor cells.

CRISPR/Cas9 Mechanism Diagram

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This diagram illustrates the CRISPR/Cas9 genome editing process. 1 shows the Cas9 enzyme bound to a guide RNA (gRNA) targeting a specific DNA sequence. 2 indicates the double-strand break made by Cas9 at the target site. Following the break, two repair pathways are possible: 3A, Nonhomologous End Joining (NHEJ), which often results in frameshift mutations leading to a “knock-out” of the gene. Alternatively, 3B, Homology-Directed Repair (HDR), uses a provided donor DNA to precisely insert or replace a sequence, resulting in an “edited sequence” or “knock-in”.

Southern Blot Procedure

1. DNA sample is enzymatically cleaved into smaller pieces, which are separated on a gel by electrophoresis, and then transferred to a membrane.

2. Membrane is exposed to labeled DNA probe that anneals to its complementary strand.

3. Resulting double-stranded, labeled piece of DNA is visualized when membrane is exposed to film or digital imager.

Other Blotting Techniques

Northern blot

Similar to Southern blot, except that an RNA sample is electrophoresed. Useful for studying mRNA levels, which are reflective of gene expression.

Western blot

Sample protein is separated via gel electrophoresis and transferred to a membrane. Labeled antibody is used to bind to relevant protein.

Southwestern blot

Identifies DNA-binding proteins (eg, c-Jun, c-Fos [leucine zipper motif]) using labeled double-stranded DNA probes.

SNOW DRoP Mnemonic

Southern = DNA

Northern = RNA

Western = Protein

Northern blots detect splicing errors.

Southern Blot: Pedigree and Results

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This image displays a pedigree chart alongside Southern blot results. The pedigree shows two parents and their children, with genotypes indicated as AA, Aa, or aa. The Southern blot panel below correlates with these genotypes, showing distinct banding patterns for mutant and normal alleles. Individuals with the ‘AA’ genotype show only the normal band, ‘aa’ individuals show only the mutant band, and ‘Aa’ heterozygotes display both normal and mutant bands, indicating the presence of both alleles.

Topics Covered:

• CRISPR
• Cas9: Introduction and Mechanism
• Visualizing CRISPR
• Cas9 Repair Pathways
• Steps of Southern Blotting
• Comparing Blotting Methods
• Interpreting Southern Blot Results