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Providing Advanced Biological Technical Services for Researchers in Fundamental Science
Gene Knockout Service for Prokaryotes
High-Efficiency CRISPR-Cas9 Gene Knockout in E. coli and Common Prokaryotic Hosts
The CRISPR/Cas system can integrate genetic material from invading bacteriophages into one or more CRISPR loci within the bacterial genome, serving as a permanent "memory." When the bacterium is reinvaded, these CRISPR loci are transcribed into CRISPR RNAs (crRNAs). The crRNAs then guide the Cas9 endonuclease to cleave the invading foreign nucleic acids based on sequence complementarity.
Genome editing technologies derived from the CRISPR/Cas system have been engineered into a two-component system consisting of a guide RNA (a chimeric RNA combining crRNA and tracrRNA, known as single-guide RNA or sgRNA) and the DNA endonuclease Cas9. This sgRNA-Cas9 complex can be directed to specific DNA sequences, enabling gene knockout, knock-in, or site-specific modifications. CRISPR-Cas9 has been widely applied in various fields, including genome editing and nucleic acid detection in living cells.
Technical Elements
Custom-designed broad-host-range CRISPR vectors
High-activity Cas9 protein variants optimized for prokaryotes
Precision-designed sgRNAs targeting critical genomic regions
Technical Advantages
High Editing Efficiency: For common prokaryotic hosts such as E. coli, gene knockout efficiency exceeds 90%.
Low Off-Target Effects: sgRNA sequences are precisely designed using databases to minimize off-target effects.
Broad Host Compatibility: Compatible with widely used strains including MG1655, Stbl3, DH10B, Stable, and BL21(DE3).
Comprehensive Technical Support: From target design to troubleshooting, our expert team offers full support including experimental planning, data analysis, and problem-solving at every critical step.
Sample Acceptance Criteria
Sample Type | Submission Requirements | Shipping Conditions |
|---|---|---|
Sequence for Gene Synthesis | None | None |
Constructed plasmid / Intermediate vector plasmid | Concentration > 100 ng/μL, volume > 20 μL; provide vector sequence and antibiotic resistance information; free of contamination. | Ice pack |
Bacterial culture (with plasmid or vector) | Volume > 500 μL; provide vector sequence and antibiotic resistance information; no contamination | Dry ice shipping: 1 day (5 kg), 2–3 days (10–20 kg), 3–5 days (20–30 kg) |
Plate culture | Contamination-free | Room temperature |
Service Process and Timeline
Service Process | Working Days | Deliverables |
|---|---|---|
Customer Order Placement | 10 | 1. Glycerol stock and plate culture of successfully knocked-out strains |
Gene Synthesis / Plasmid Verification | ||
CRISPR Knockout Target Design | 30 | |
Knockout Module Construction | ||
Vector Construction and Plasmid Extraction | ||
Chemical / Electroporation Transformation | ||
PCR Verification | ||
Sequencing Confirmation | ||
Positive Clone Amplification | ||
Result Validation |
Frequently Asked Questions
Q: What are the differences between CRISPR/Cas9 gene knockout and RNAi?
A: In terms of gene expression suppression efficiency, RNA interference (RNAi) reduces gene expression at the post-transcriptional level, whereas CRISPR/Cas9 targets DNA directly and can permanently alter or knock out gene expression. In some cases, complete knockout via CRISPR/Cas9 is preferred to avoid any residual low-level protein expression that might cause unintended effects.
As for off-target effects, the CRISPR system typically requires targeting regions near the transcription start site to be effective, which significantly reduces its off-target rate compared to RNAi.
Case Project Results
Transformation Efficiency Analysis: Electroporation produced dense and healthy single colonies, indicating successful transformation.
PCR Validation Result: The target gene was successfully knocked out.
References
Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cassystems. Science. 2013;339(6121):819-823. doi:10.1126/science.1231143
Ready to Start Your CRISPR Knockout Project?
Partner with ProNet Biotech to achieve precise and efficient gene knockout in E. coli and other prokaryotic strains. Whether you aim to target a single gene or building complex mutant libraries, our expert team is here to support you at every step.
Contact us today for a free consultation or request a custom quotation.
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