The rapid evolution of the CRISPR-Cas9 gene editing field has led to a proliferation of technology formats and applications that can seem intimidating for the first-time researcher. How can you be sure you are properly addressing your research concern? What do you need to do to prepare for your experiment? How can you best ensure your success? Below is a quick list of the 5 main points one should consider when planning a gene knockout experiment with CRISPR-Cas9:
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Know your target
What gene are you interested in? Is there a specific region you would like to target? Due to the fact that not all cleavage events will cause full knockout of the gene of interest, it is recommended to use multiple (3-4) guide RNAs that target different regions of your gene. Using guide RNAs that have been designed using a functionality algorithm, such as that used for predesigned Edit-R guide RNAs, is one way to increase your chances of success without any cumbersome design steps. -
Choose your Cas9 nuclease
Cas9 nuclease options are split into two main types: vector-based and DNA-free (protein, mRNA). Factors that can influence your decision are whether you want to enrich for Cas9 expressing cells, wish to establish a stable cell line, or prefer to avoid the potential for vector DNA integration into your genomic DNA. Here is an article that describes these factors in more detail. -
Select a guide RNA (gRNA)
The two RNA components of the CRISPR genome editing reactions, CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA), may consist of a chimeric single guide RNA (sgRNA) or a two-part synthetic oligo system. Lentiviral sgRNA vectors are convenient for pooled screening applications, difficult-to-transfect cells, or if enrichment of a population is required. Chemically synthesized crRNA:tracrRNA carries the advantage of time savings (arrive ready-to-use, no need for vector cloning steps), the versatility to incorporate chemical modification patterns, and the opportunity to set up an editing system free from risk of incorporation of exogenous vector DNA[1]. -
Consider cell population vs. clonal selection requirements
For many single-gene knockout applications, a clonal selection workflow is considered best practice to ensure a homogenous population with a fully characterized loss-of-function mutation for the gene of interest. However, when performing a CRISPR screen targeting multiple genes at once, this may not be feasible. In such cases, consideration should be given to the phenotypic assay and whether it is robust enough to detect knockout in a mixed population. -
Detect your knockout
Once editing is accomplished, what is the extent of the detection assay required? For screening purposes, a phenotype can be the indicator of success. However, for most purposes, molecular techniques will be needed to more definitively characterize the editing event. DNA mismatch detection assays such as T7EI or Surveyor are typically used as a first pass to verify the CRISPR-Cas9 reaction resulted in insertions and deletions (indels) in the initial mixed population of cells. However, Sanger sequencing is required for ultimate verification of knockout of all alleles in a clonal cell line, and western blot analysis can provide an estimation of remaining protein levels. See an example in the Gene Editing Workflow Guide.
References
- Kelley, M.L., Ž. Strezoska, et al. Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing. J. Biotechnol. 233, 74-83 (2016). doi:10.1016/j.jbiotec.2016.06.011