With the use of CRISPR knock-out (KO) cell lines on the rise, many researchers are exploring how to generate cell lines in their own labs. Due to the significant time investment and high level of technical skill required, generating your own cell lines can often be a frustrating trial-and-error process where failure is invisible until the end. Automation and large-scale CRISPR operations can help to optimize your protocol¹, but many labs aren’t equipped to deal with such extensive operations.

What is CRISPR-Cas9?

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences that make up the bacterial adaptive immune system. This protects bacteria against and destroys invading viruses by providing repeating spacer sequences of target DNA for nucleases to cut². Transcribed CRISPR sequences guide the Cas9 enzyme to cleave DNA that disables and disrupts viral genomes at specific locations.

The molecular components of CRISPR-Cas9 can now also be used as a gene-editing system in eukaryotic cells³. This system uses short synthetic stretches of guide RNA to direct Cas9 enzymes to a target location in the genome. Once in place, Cas9 cleaves the DNA three bases from the end of the target sequence, thus providing a precise replacement, deletion, or insertion of genomic material. In this way, a portion of the gene (a small to large deletion) can be excised to knock out the gene function, or mutations that cause disease can be replicated to provide models of gene-based disorders.

Why use CRISPR-Cas9 technology for generating cell lines?

CRISPR-Cas9 technology has many benefits for gene editing and cell line creation:

• It is three to four times more efficient than traditional systems like ZFN and TALEN⁴.
• Multiple genes can be modified easily by introducing several different guide RNAs⁵.
• Target genes can be modified precisely during knock-out cell line creation, resulting in complete ablation of the protein it encodes.

However, various elements need to be optimized to successfully produce a precise CRISPR-Cas-9 knock-out.

Where cell line production can go wrong in a basic CRISPR-Cas9 protocol

The average time researchers take to generate a CRISPR knockout (KO) cell line is almost five months, and most researchers are forced to restart their experiments three or four times before they achieve the knock-out cell lines they need³.

The standard workflow to generate CRISPR KO cell lines involves five major steps. The most common challenges arise for researchers during the first five stages of this process, especially during transfection, enrichment or isolation and expansion.

1. Knock-out guide design and production

A wide variety of guide RNA formats can be used to generate CRISPR KO cell lines; single guide RNA (sgRNA), plasmid, and lentivirus. Each of these formats presents unique challenges.

2. Transfection

Determining the most efficient method for cell transfection is considered the most difficult step in a CRISPR workflow by many researchers¹. Transfection conditions require extensive optimization for each cell type in order to deliver gene-editing machinery into the cell accurately.

Stem and primary cells and immune and hematopoietic cell lineages are more difficult to transfect as they have altered cell repair mechanisms and lower cell viability. Successful transfection often depends on your previous experience and understanding of the cell characteristics within  your cell line of choice.

3. Enrichment, single-cell isolation and expansion

Though not essential, clonal selection is the best practice for developing a homogenous cell population containing the desired knock-out. Enrichment reduces the number of required cell passages to achieve a clonal population, increasing the health of the cell population in downstream experiments.

However, these stages can be time-consuming and technically demanding, requiring extensive screening of multiple clones to identify the required knock-out. Without fully optimized growth conditions, it can lead to the death of precious cell samples and, therefore, the loss of all work during this process.

4. Confirmation of edits

Once a CRISPR KO cell line has been established, adequate validation of the specific gene edits is important. To ensure downstream viability, this usually requires characterization of your knock-out. This can be done with a phenotype as an initial indicator, but molecular techniques are also needed to definitively characterize the clone. DNA mismatch detection assays can also verify CRISPR-Cas9 reactions at first pass, but full DNA sequencing is essential to verify the knock-out of all alleles without unwanted deletions.

How to validate your CRISPR knockout

Validating via multiple methods offers the best assurance of an accurate gene edit. Common methods to validate engineered cell lines include Sanger sequencing, next-generation sequencing, and qPCR to verify the edit at a genomic level.

Western blot and mass spectrometry can provide confirmation of the KO at the proteomic level.

For functional studies, immunohistochemistry, immunocytochemistry and FACS are often used.

Find out more about Abcam's ready-made KO cell lines

References

1.      Synthego. CRISPR Benchmark Report (2019)

2.      Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).

3.      Lomov, N. A., Borunova, V. V. & Rubtsov, M. A. CRISPR/Cas9 technology for targeted genome editing. Biopolymers and Cell 31, 243–248 (2015).

4.      Ye, L. et al. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. Proceedings of the National Academy of Sciences 111, 9591–9596 (2014).

5.      Kabadi, A. M., Ousterout, D. G., Hilton, I. B. & Gersbach, C. A. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Research 42, (2014).