Custom Cell Line Engineering




QuestionAnswer

Do you usually use drug selection? 

Drug selection is used when necessary and as standard we would use G418 selection, but can use alternatives such as hygromycin B or puromycin if required.

Can the antibiotic resistance be removed at a later date? 

Absolutely.  All selection markers are flanked by LoxP sites allowing cre-mediated excision to be used to remove the marker leaving only a single LoxP site (34 bp) as a scar.  This step could be performed either by the researcher when necessary or as a subsequent project by Horizon. 

What do you do to validate the gRNAs in the cell line?

We validate all gRNAs in a test cell line, usually, HEK-293.  gRNA and Cas9 expression plasmids are transfected into the test cell line; 48-72h later cells are harvested and a Surveyor assay is used to assess cutting frequency. The most suitable gRNA is chosen based on cutting frequency alongside suitability for targeting (based on location with respect to the desired mutation)

Can you design gRNAs to exons and introns?

Yes, gRNAs are designed to exons or introns depending on what is more appropraite for the project. For knockout projects we aim to design gRNAs to conserved exons in the first third of the gene.  For knock-in projects where a donor is required to introduce a change in the coding sequence, gRNAs are designed  near to the required change in the exon.  For knock-in donors, if a gRNA is designed to an exon, in order to prevent re-cutting of the targeted allele, we may also introduce silent mutations in the donor to disrupt the gRNA target site/PAM site.  However, depending on gene structure, it is also possible to site the gRNA in the intron distal to that in which, for example, an antibiotic resistance marker is located.  While this would have an impact on targeting frequency, and would require modification of intronic sequence to prevent re-cutting of the targeted allele, it would mean that exonic silent mutations would not be required.

Do you ever use more than one gRNA in parallel as a back-up?

We aim to choose a gRNA where validation in a test cell line indicates high cutting rate.  However, should no such gRNA be identified, alternate options could be pursued includeing design of more gRNAs oruse of multiple gRNAs in parallel.

How do you assess the level of targeting? 

After antibiotic selection, the resistant pool of cells can be assessed for targeting in two ways.  Firstly a Surveyor assay can be used to assess rate of Cas9 activity.  Secondly, PCR based screening primers are used to assess presence of on target integration of the donor plasmid in the pool in a semi-quantative way.  These two measures allow us to assess likelihood of project success before screening of clonal populations takes place.

How many single wells do you try and grow up in one round?

This is dependent on the targeting project and likely targeting rates. If we are anticipating a relatively low rate of delivery of bi-allelic targeted clones, we will be aiming to grow between 300-500 clonal populations over 20x96 well plates.

How many clones do you initially screen? 

This will depend on the results from assessment of targeting at the pool level, but will likely range from 100-500 clones.

In the case that I provide a cell line for targeting, is there anything else that I have to supply for any step of the process? 

No, other than any information you have regarding required culture conditions for the cell line, and any other information which may be helpful to us such as suitable conditions for transfection, single cell dilution, etc, if available.  On receipt of a new cell line the team will assess the cell line for its suitability for gene editing (Cell line characterisation). During Cell line characterisation we assess the ability of a cell line to tolerate single cell dilution, determine optimal transfection conditions and carry out some genotyping assessments to determine copy number and and any sequencing of the target locus.  The next phase of the project is reagent design. On completion of reagent design, we will provide a summary slide documenting the design of the reagents,  we ask that you sign-off to agree that you are happy with the design before we proceed to manufacture.  Aside from this, we will provide regular updates on project progress by email, and where necessary or desired (e.g. at key decision points) also schedule calls to discuss project progress.

Can you make a homozygous knock-in of a point mutation in a diplod cell line?  What happens if no homozygous knock-in clones are recoverd?

Homozygous knock-in of a point mutation can be made using CRISPR plus a donor strategy.  If however, homozygous knock-in of the mutation was not achieved, an alternative genotype which may be able to be supplied, for illustration GENE1 (knock-in/null) where the second allele was knocked-out by Cas9 activity followed by non-homologous end-joining mediated repair, leading to an out of frame insertion or deletion.  The outcome of a (knock-in/null) genotype the generation of a cell line that only expresses the protein containing the mutation.

if I requested a homozygous knock-in and only a heterozygous mutation was produced, would Horizon be able to determine the extent of mutant vs wt expression?

Assessment of mutant vs WT expression at the RNA level is something we carry out as standard as part of every targeting project (where applicable).  We use both standard RT-PCR followed by Sanger sequencing, and quantative droplet digital PCR (ddPCR) to assess level of expression.  However, we do not guarantee to be able to deliver clones which express the mutation at a particular level. 

How do you assess CRISPR-engineered cell lines for off-target editing? 

This has two elements.  
1) off-target Cas9 mediated NHEJ 
2) As the majority of our projects are KI, assessment of the off-target integration of the exogenous donor sequence.  
Assessment of off-target NHEJ is an optional service and can be adjusted to suit the requirements of our clients.  Ranging from assessment of the top 5 predicted off-target sites by Sanger sequencing through to genome sequencing.  The price of a project reflects the degree to which a client wishes to investigate this.

Assessment of off-target donor integration can be performed by several methods depending on the type of donor being utilised and the most appropriate strategy is deployed for a given project.

Do you have a general strategy for making Kis?  And do you use antibiotic selection markers in your donor designs

We have established a number of different strategies that can be deployed to generate a KI model.  Although all follow a generalised project profile, we evaluate the gene/mutation target (copy number and likely biological impact), the type of modification required (conditional approach, reversion, mutation introduction), the cell line of interest, the end use of the cell line as well as any specific requirements/limitations of the client in order to propose the final strategy.

We have had success using both AAV and plasmid donors with antibiotic selection cassettes, as well as oligonucleotide donors.  We choose the most appropriate strategy based on these factors, and if required can thoroughly discuss the merits of different approaches with the Client prior to making a decision on which strategy to use.

Do you think the technology is going to be applicable to primary cells/cells with limited proliferative capacity?

We haven't performed such engineering, but there are papers in the literature that indicate that Cas9/sgRNA could be introduced to terminally differentiated neurons with lentiviruses or biolistic delivery and that editing rates were claimed to be very high.  However, given the limited proliferative capacity this would be a very different type of engineering project than generating a clonal, precisely engineered cell line.  We are considering this area and have begun scoping out requirements but cannot give any firm info at this point.

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