CRISPR is opening up a wealth of opportunities for gene editing, but maintaining authenticity is proving to be a big challenge, explains Isobel Atkin, Senior Portfolio Manager at Ximbio,

In our previous blog, we discussed how the exciting gene editing technique of CRISPR has attracted attention for what, to many researchers, might seem the wrong reason – it’s the subject of a prolonged dispute over patents. Accounts in the mass media have focused on controversial applications to genetic engineering of human beings including, potentially, germline modification.

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Most researchers, however, are excited about CRISPR because it is the quickest and cheapest method of genome editing, costing only around a hundred pounds and a few weeks of time. CRISPR also requires less expertise in molecular biology, because the key step is designing the guide RNA that directs the nucleases to the cutting sites. With CRISPR, researchers can create new cell lines, mouse models and other systems to study and analyse the biology at a molecular level.

These developments present both challenges and opportunities for an organisation such as Ximbio whose portfolio consists of antibodies, cell lines, mouse models, plasmids, small molecules, and other research tools with the aim of sourcing and sharing such reagents within the life sciences. If it’s now cheaper and quicker to create cell lines using CRISPR, will researchers prefer to do so in their own laboratory rather than sourcing already edited cell lines from elsewhere?

Many cell lines already exist that derive from a single individual but in different versions – for example, cancer cell lines that have been exposed to and developed resistance to different drugs. Quality control, in terms of maintaining the correct identity and avoiding cross-contamination, can be achieved by physical segregation: never having more than one cell line in a cell culture cabinet at the same time.

Dr Edward Burnett, Head of Scientific Development at the Culture Collections of Public Heath England advises: “At our European Collection of Authenticated Cell Cultures (ECACC), when making cell banks, we will not have cell lines derived from the same individual in the lab at the same time, let alone the same cabinet or incubator.”

It is this attention to detail that ensures that different lines – and different lines of the same parental origin – do not get mixed up. Quality control such as this is crucial to creative science, but it is not particularly creative science in itself and, in academic laboratories doing their own thing, there may not be the time or money to carry out this work in the necessary detail.

With traditional cell lines, authentication itself is done by checking short tandem repeats (STRs) at different loci in the genome – it’s what they use in criminology for forensics. It’s been well worked out that 18 loci are enough to ensure uniqueness to that originating individual and accommodate issues of genetic instability that can occur in immortalised cell lines. This STR profiling method is recommended by the International Cell Line Authentication Committee (ICLAC).

However, that technique is not sufficient to distinguish between two cell lines derived from the same individual if a part of the genome has been edited outside of the regions being analysed. If modern techniques are offering precision ‘surgery’ of DNA, then the analysis needs to be precise also.

 

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CRISPR can generate lots more cell lines from the same parent cell line but, because of the way in which they have been modified, there is a burden further downstream – how to check which is which. Sequencing and restriction enzyme cutting of PCR products localised to the edited region are the most commonly used methods to check that a specified part of the genome has the modification of interest; that may prove expensive, if it is to be done for every batch of a cell line made.

Sequencing of the edited genome region needs specific primers generated for each location rather than a universal test. There is also the question of off-target edits CRISPR could inadvertently produce. So, the region you are interested in has been edited as expected but have any other areas been affected? What can smaller laboratories do if they don’t have easy access to whole genome sequencing?

Checking if results are reproducible is key to firm up findings. Editing and producing a number of clones using different guide RNAs to target the same region and using different guide RNAs to different parts of the same gene are unlikely to give the same off-target edits.  Testing the hypothesis using an alternative approach to see if results concur is another.

When asked: ‘How well are people dealing with CRISPR in relation to verifying the edit and cell line authentication?’, Michael Howell, Head of High Throughput Screening at the Francis Crick Institute, said: “Very badly – the complexity is often not appreciated.”

Michael continued: “Simple phenotype variations occur with single-cell cloning. What represents average in a cell population? A single clone is just one manifestation of many possible cell phenotypes for any one genotype. If you make a knockout cell line, you can’t say this is the definitive phenotypic representation of the knockout for that particular gene of interest. Lots of properties can vary between clones.”

“For example, we used single-cell cloning to generate multiple clones from the same population and monitored their growth profiles. The growth rate of the clones varied widely, and this variation is on top of any unintended off-target edits elicited by the CRISPR reagent used.”

Michael recommended that, as a minimum for CRISPR knockout cell lines, to check the edit has actually been made, a test by western blot should be made to check loss of protein expression. This is because there are cases where, at the DNA level, the edit has been made but there is still presence of RNA and protein because of alternative splicing or initiation sites.

Michael added: “Perhaps the best proof of the link between the gene edit and the new phenotype observed would be to repair or reverse the edit – again, using CRISPR to knock-in the wild type sequence – and see that the phenotype reverts; non-trivial, but the closest thing to a gold standard verification we have.”

All these issues are already there, as a result of previous genome modification techniques, but the cheapness and speed of CRISPR does mean that the volume of cell lines that researchers are able to produce will increase and, with it, the volume of work involved in checking and maintaining authenticity goes up also. 

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