You have found a repressor protein (leuR) that prevents synthesis of leucine in Bucky anonymous. You want to knock out this gene so that leucine synthesis is unregulated. Which of the following methods would have the highest chance of success? You have the entire DNA sequence of B. anonymous.

Question

BlackTom AI · Accepted Answer

CRISPR-Cas9 enables precise, targeted disruption of a chosen bacterial gene by creating a double-strand break at a specific genomic locus, so designing a guide RNA (gRNA) that directs Cas9 to leuR is the most direct way to abolish its repressor function.

1) Relevant knowledge points and concepts
- CRISPR-Cas9 uses a ~20-nt gRNA sequence adjacent to a PAM (e.g., NGG for SpCas9) to target a specific genomic site.  
- In bacteria, CRISPR editing is typically paired with a repair template (homology-directed repair/recombineering) because double-strand breaks are often lethal unless repaired.  
- Loss-of-function in a repressor (frameshift, early stop, deletion) should derepress the leucine biosynthesis operon, increasing leucine synthesis.  
- Verification methods: PCR and Sanger sequencing of the locus, and biochemical assays for leucine (HPLC, LC-MS, or growth assays).

2) Step-by-step reasoning and strategy
- Using the complete genome sequence, identify the leuR coding sequence and surrounding region.  
- Select candidate 20-nt target sites within an essential region of leuR that are adjacent to a PAM (NGG for SpCas9). Prioritize sites near the start codon to maximize chance of loss-of-function.  
- Perform in-silico off-target analysis across the genome to minimize unintended cuts.  
- Design a repair template that introduces a short deletion or premature stop (or frameshift) and includes homology arms (~500–1000 bp depending on species and system) for recombineering.  
- Clone the gRNA and Cas9 (or use a two-plasmid system) and deliver to B. anonymous (electroporation, conjugation). Provide the repair template concurrently. Use a counterselection strategy if needed.  
- Screen colonies by PCR across the leuR locus and Sanger sequence to confirm the designed edit.  
- Assay leucine production (HPLC/LC-MS, or growth in leucine-limited medium) to confirm derepression.

3) Intermediate steps shown above: target selection (20-nt + NGG), repair-template homology arms, PCR/sequencing confirmation, biochemical assay.

4) Why this is correct
CRISPR offers specificity (guided by genome sequence) and the ability to create a defined knockout that eliminates repressor function. With the genome known, off-targets can be minimized and the repair outcome precisely engineered to ensure unregulated leucine synthesis.

5) Why other methods are less favorable
- Random transposon mutagenesis: may disrupt leuR but is random, can cause polar effects, and requires extensive screening.  
- Classical homologous recombination without CRISPR: possible but slower and less efficient because you lack the selectable double-strand-break counterselection CRISPR provides.  
- Antisense/RNA interference: bacteria generally lack RNAi and antisense approaches give knockdown not reliable permanent knockout.

6) Conclusion
Designing a guide RNA and using CRISPR to knock out leuR, then screening for increased leucine production, is the best method. This matches the provided correct answer.

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