Design primer for CRISPR-Cas9 system?

Certainly, validating gene knockout following CRISPR/Cas9 editing is a critical step to ensure the accuracy and efficiency of the gene editing process. Here is a systematic approach for validation:

Polymerase Chain Reaction (PCR): Conduct PCR with primers flanking the CRISPR target site. By comparing the PCR products from edited and unedited cells, one can detect insertions or deletions (indels) at the target site, which are indicative of gene editing.

Sanger Sequencing: Sequence the PCR products using Sanger sequencing. This method allows for direct visualization of the mutations introduced at the target site, including the specific indels responsible for the knockout.

T7 Endonuclease I Assay: This assay is employed to identify mismatched DNA strands, which are indicative of indels. T7 Endonuclease I cleaves these mismatches, and the resulting fragments can be analyzed through gel electrophoresis.

Surveyor Nuclease Assay: Similar to the T7 assay, this method detects indels by hybridizing mismatched DNA followed by digestion with Surveyor nuclease, with the products analyzed on a gel.

Quantitative PCR (qPCR): Use qPCR to assess the levels of mRNA expression from the targeted gene. A significant reduction in mRNA levels post-editing suggests a successful knockout.

Western Blotting: This technique measures the protein levels of the targeted gene. The absence or substantial reduction of the protein post-CRISPR editing can indicate a successful knockout.

Immunofluorescence or Flow Cytometry: If the protein has a known cellular localization, these methods can visually confirm the presence or absence of the protein in the cells.

Functional Assays: Depending on the known functions of the gene, carrying out relevant functional assays can provide evidence of the loss of gene function. This could involve assays for cellular phenotype, enzymatic activity, or other specific functions.

Off-Target Analysis: It's also critical to evaluate potential off-target effects. Predict potential off-target sites using computational tools, followed by sequencing to confirm.

Replication and Confirmation: Replicating the experiment and confirming gene knockout in multiple clones adds robustness to the validation process.

In summary, a combination of molecular and biochemical techniques, along with functional assays, provides a comprehensive approach to validate gene knockout. This thorough validation is essential to confirm the specific and effective knockout of the target gene using CRISPR/Cas9 technology.

l Reviewing the protocols listed here may offer further guidance in addressing this issue.

Hendrik de Buhr

You might find help regarding plasmids, gRNA design and cloning here: https://www.addgene.org/crispr/

Tuyet Van

Dear Hendrik,

Thank you for your suggestion. I think it might be good for me to study more about this system.

Thank you so much.

Aem. S.S

hello everyone i am facing the same issue. please help me . i have selected some N-20 sequences depending on PAm region of my gene of interest but my question is./.

should i insert my N-20 sequence upstream of sgRNA using enzymetic ligation or just use inverse PCR system to insert my N-20 sequence in the plasmid containing sgRNA.

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