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Planning a CRISPR gene editing experiment to maximize your chances of success

Jun 10, 2016 2:10:39 PM No Comments

While CRISPR-Cas9 has made gene editing cheaper, easier and more accessible than ever before, using the system can in some cases still be challenging, and no scientist can yet be 100% certain of success. With careful preparation and planning however, chances of success can be significantly boosted.

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Figures below give an overview of the process required to generate either a knockout (KO) or knockin (KI) cell line using CRISPR.

Gene editing workflow Gene editing knockin workflow

Here's how we recommend planning for any genome editing experiment:

Start with a clear project goal

  • What is the aim of the project (knockin, knockout, loss of functional activity, etc.)?
  • Is a homozygous or heterozygous event required?
  • How much time and money are you prepared to devote to achieving your aim?
  • What would success look like? (i.e. is a homozygous knockin the only possible outcome, or will a het be sufficient?)

Select a suitable cell line and optimize your protocols

When starting an engineering project it is particularly valuable to know as much as you can about both your target gene and the target cell line. For example STR (short tandem repeat) genotyping is a valuable technique that is widely used to authenticate the ancestry of a cell line. SNP6.0 data can give valuable knowledge on the copy number of the target gene which is essential to accurately genotype the modified cell lines generated using CRISPR.

In addition, it is valuable to have accurate sequence of the target locus and in particular to confirm the presence of the gRNA target site and to locate any single nucleotide polymorphisms that may be present as these can impact gRNA binding, screening and validation of the engineering event.

Problems coming from the choice of cell line is one of the most common blocks to successful genome editing, and so when selecting a cell line it is important to address the following questions:

  • How well the cell line can be transfected or infected (transfection efficiency)?
    • Very low transfection efficiency can have a huge impact on recovering modified cells, even if tranfected cells can be enriched.
  • How well the cell line tolerates single cell dilution (i.e. generation of a clonal cell population from a single cell)?
    • Some cells do not survive single cell dilutions, and some cells remain stubbornly clumped. If the end goal is to isolate a clonal, targeted cell line then optimising this step is critical.
  • The likely impact of the modification on cell viability and growth?
    • If a gene modification reduces viability of cells, then while it may be possible to recover a targeted clone, this will affect the number of clones you have to screen to find it.
  • What is the copy number of your gene in your cell line?
    • This will impact your ability to make homozygous modifications
  • How fast do your cell grow (doubling time)?
    • Not necessarily a limiting factor when it comes to final success, but should certainly be factored into your timelines and deadlines
  • Sequence data for the target gene in the cell line to be used (versus a reference sequence).
    • Working with the sequence file from your cell line can help with selecting a guide RNA that is more likely to be effective.

For more guidance on selecting a cell line, check out our guide

gRNA design and specificity

When it comes to balancing specificity and efficiency there is a trade off. For example, using the nickase system is certainly more specific, but because you're relying on the binding of a pair of guides, this almost certainly comes at the expense of efficiency. Variables to keep in mind when planning a CRISPR experiment include:

  • Proximity of the intended cut to the desired mutation (generally the closer the better, but not always possible)
  • Off-target potential.
  • Which Cas9 is more suitable for use, Cas9 wild-type or Cas9 nickase?

As cell biologists we have the luxury of designing multiple independent guides, and isolating multiple independent clones and so the risk of off-target cutting can be controlled in this manner. As such if you're just starting out with CRISPR, my recommendation would be to prioritise efficiency in the first instance whilst you're getting the process up and running in your lab, and consider bringing in more specific systems after achieving success.

Here's our advice on choosing guide RNAs

gRNA activity

It's been our experience that a high proportion of guides are "active" - but it is still good practice to design multiple guides (2-3) and test before hand. Quantitation of guide efficiency can also provide an idea of how many clones will need to be screened to recover a modified one.

Check out our guide to testing guide activity.

gRNA delivery

As mentioned above, delivery of CRISPR reagents can actually be a big stumbling block to successful gene editing. It's well worth spending some time optimizing your transfection or infection protocols for each cell line. Make sure you are working with the absolute best route to introduce gRNA, Cas9 and donors into your target cell line. If you're doing a knockin, it's also worth answering the following questions.

  • What type of donor is most suitable (oligo, plasmid, AAV, Adenovirus)?
  • What is the best sequence design for the donor?
  • Should selection be used?

Screening for modified clones

Factoring a screening strategy into your experimental design can save a significant amount of time at the bench. At Horizon, we will where possible introduce or disrupt a restriction site as part of the genome editing to allow us to use restriction fragment length polymorphisms (RFLP) when screening. Some labs use cutting with the gRNA and Cas9 itself as a screening tool.

Cell line validation

The purpose of screening is to identify those clones that have undergone some kind of disruption - it is possible they are heterozygous or homozygous, and the nature of the modification will not be known. It is important therefore to validate the individual clones and confirm which modifications have actually occurred at the target site. i.e.:

  • Is it a heterozygous or homozygous clone
  • Are the modifications as expected/desired (frameshifts, knockins)
  • Have off target events occured?
  • Does genetic drift need to be considered?


Even with all these steps planned for, isolating targeted cell lines can still be challenging, especially for example if you're performing knockins. In these instances, where it can often just become a numbers game to screen sufficient clones to isolate the right one, then making sure you're working in optimal conditions can make the difference between hundreds of clones and thousands.

#Gene editing

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