How to Validate a CRISPR Knockout

Innovations in gene editing are rapidly revealing relevant applications in a wide array of industries. From animal breeding and agriculture to disease therapeutics and human health research, the manipulation of genes results in the advancement of scientific knowledge with great societal impact.

One specific, well-known method to edit particular genome sites is CRISPR/Cas. Scientists can easily and effectively use CRISPR technology to modify genes by, for example, knocking out genes and then observing the altered functioning. This blog post will highlight the importance of the CRISPR technology and how to validate a CRISPR knockout for success.

The Value in CRISPR Knockout Validation

 
The repetitive palindromic sequences that make up CRISPR prove to be a straightforward target for researchers to remove from the genome using Cas9 nuclease as an instrument to cut the target DNA site. When the genetic material repairs itself, it can result in missing bases and thus a CRISPR knockout.

 
A CRISPR knockout can then be observed for altered gene function for desirable results in test subjects. For example, recent breakthroughs using CRISPR knockout techniques include detecting viruses in blood, removing defects from different diseases, and increasing plant resistance to disease.

 
The great significance of these gene editing experiments supports the value in CRISPR knockout validation. While CRISPR is a very useful technology, the intended gene edits are not always guaranteed. Performing checks and verifying whether a CRISPR knockout was successful is crucial to avoid the immense loss of time and cost of performing these innovative laboratory strategies. Validation of a CRISPR knockout ensures proper methodology, accurate results and greenlights the next steps of an experiment, which include monitoring altered genetic functions in the cell.

CRISPR Knockout Validation by PCR Sequencing and Protein Expression Measurements

 
Proper verification of CRISPR knockout results allows researchers to alter and correct their techniques accordingly to improve precision and avoid error. Cutting-edge strategies have been developed to answer the question of how to validate a CRISPR knockout. PCR sequencing is a primary method of gene targeting confirmation.

 
This is conducted by performing PCR on amplified, extracted genetic material from both edited and control cell populations and running them on gel to then perform PCR product size comparisons.

 
Sangar DNA sequencing is a reliable, sensitive and precise PCR procedure that can identify genetic mutations induced by the CRISPR knockout. If a validation measure requires increased sensitivity, a TIDE (Tracking of Indels by Decomposition) Assay could be utilized where DNA is amplified from edited cells and then Sangar sequencing is performed and analyzed by advanced software.

 
When knocking out a gene, the levels of protein expression should be altered and thus measurements of protein expression can also be used to validate a successful CRISPR knockout further. This can be accomplished by the Western Blot technique or by mass spectrometry. Mass spectrometry is a popular and dependable verification method that relies on the isotopic labeling of proteins which are then differentiated and quantified.

 
Following the validation of a CRISPR knockout, the impacts of the modified cell in experimental systems can be measured and analyzed. Other, less popularly used assays also exist for specific verification conditions.

Validating CRISPR Knockouts with Advanced Proteomics at Biognosys

 
Experts at Biognosys have enabled effective and successful validation of a CRISPR knockout using a quantitative proteomic approach. Able to simultaneously discover and analyze protein modifications, proteomics is valuable in learning more about gene expression and function. A leader in mass spectrometry proteomics, Biognosys presents advanced solutions addressing how to validate a CRISPR knockout. With these innovative products and services, Biognosys facilitates breakthroughs in disease prevention. To find out more about advanced proteomics, contact us today.