A Guide to CRISPR Cell Line Editing
– Gene Knock Out Workflow

In this guide we will outline an automation led, streamlined pipeline for the
efficient generation of knockout mammalian cell lines developed at Oxford Genetics.

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Part One – Cell Line Characterization and Target Gene Copy Number Analysis

 

 

A cell line engineering project often begins with the choice of which cell line you want to modify. The typical sources for this can be an existing line from your own laboratory, or a commercially available cell line from a repository such as ATCC or ECACC. The challenge with using an existing line is confirming its identity.

It has been estimated that between 15 and 20% of the time, cells used in experiments have been misidentified or cross-contaminated with another cell line. This often makes obtaining your cell line from a repository a better choice. At Oxford Genetics as an early quality step and to determine the identity of the cell line Short Tandem Repeat (STR) analysis is performed.

Once the cell line's identity has been confirmed, it needs to be assessed for its growth kinetics, its ability to expand from a single cell and its response to various transfection methods. At Oxford Genetics, cell characteristics are determined using a robotics driven automated platform to ensure rapid and consistent analysis of individual cell lines. Example data from the cell tracking software is shown below in part 4. This fully automated process includes routine cell feeding, hit picking of confluent clones and subsequent clone expansion allowing for high throughput determination of cell growth kinetics.

It is also vital in the early stages of any gene editing experiment to determine the number of copies of the target gene present within the cell line. Determining the copy number of target genes is achieved using data from multiple sources including quantitative PCR (qPCR) and next generation sequencing (NGS).

 

 

 

 

 

Part Two – sgRNA Design and Determination of Cas9 Delivery Method

 

 

Part one has determined cell line identity, target gene copy number and that the selected cell line is appropriate for the intended gene edit. The components of part two are design of the sgRNA and determination of the best approach to deliver Cas9 and sgRNA to the cells. There are numerous algorithms available for designing sgRNAs. At Oxford Genetics our approach is unique.  We use the latest proprietary software in sgRNA design to maximize targeting and minimize off target effects. This design algorithm capable of delivering sgRNAs for as many target genes as you require. For more information view our high fidelity CRISPR libraries

Once the guide sequence has been selected the next choice is what delivery method to use. Here, Oxford Genetics has flexibility in it's approach. Options include plasmid DNA, lentivirus and Cas9/sgRNA ribonuceloprotein (RNP) complex. The method of Cas9and sgRNA delivery should be selected with consideration to the cell line, with each method having unique advantages (Table 1).

 

 

 

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Cas9 transfection efficiency can be assessed by multiple methods including fluorescence microscopy (Figure 1). Cas9-gRNA is delivered to cells via an optimized method of lipid-based transfection or electroporation. Transfection efficiency is subsequently validated upon visual inspection under the microscope using imaging software.

 

 

 

 

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Part Three – Validation of Cas9 delivery and sgRNA targeting

 

 

Having designed the sgRNA and determined the optimal Cas9 delivery, the next step is to validate both Cas9 and the subsequent targeting of the Cas9 enzyme to the cut site by the sgRNA. The presence of Cas9 in target cells can be confirmed by fluorescent microscopy.

Within a heterogenous population of edited cells, the edit of the target gene can be confirmed by performing a T7 endonuclease assay (Figure 2). The T7 assay can accurately recognize insertions and deletions that are a result of non-homologous end joining (NHEJ) activity. A sample of the edited cell population can be used as a direct PCR template for amplification with primers specific to the targeted region.

The PCR product is then denatured and reannealed to create heteroduplex mismatches where double-strand breaks have happened, resulting in insertion/deletion (indel) introduction. These mismatches are recognized and cleaved by T7, and the cleavage is easily detectable by agarose gel analysis (Figure 2).

 

 

 

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Part 4 – Single Cell Sorting of Edited Populations

Having confirmed both the presence of Cas9 and the specific editing of the target gene transfection individual edited cells need to be sorted to form individual clonal populations. This can be achieved by simple serial dilution, but utilization of FACS enrichment and single cell sorting of Cas9 positive population is more efficient. An example of this single cell sorting is shown in Figure 3.  



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Individual cells are expanded to produce clonal populations of cells (Figure 4). Figure 4 shows the expansion of a single CRISPR edited cell to clonal population of cells using an automated platform for tracking cell growth.The fully automated process of routine cell feeding, hit picking of confluent clones and subsequent clone expansion used at Oxford Genetics allows for high throughput and efficacious genetic engineering of mammalian cell lines. 



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Part Five – QA of Clonal Populations

 

 

Following expansion of the clonal population, samples undergo NGS to confirm the genomic modification. Analysis of the NGS data from Oxford Genetics automated cell line engineering platform reveals a highly consistent pattern of editing even across as many as 48 different samples (Figure 5). NGS validation of indel formation is standard practice and can be complemented by various proteomic or immunoblotting analyses for loss of protein.

 

 

 

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The information above is representative of the Oxford Genetics gene editing service offerings. Each stage of the workflow has been optimized and quality-assured for high throughput and large-scale demands, but is also flexible and can be adapted to more specialized, small-scale projects.

All steps of the process are validated to highest industry standards ensuring confidence in the validity of your model.

 

 

 

Interested in starting a CRISPR project with us?

 

 

 

Start your project

Why work with Oxford Genetics?

•Each step of the gene editing workflow has been optimised and quality-assured for high throughput and large-scale demands, but is also flexible and can be adapted to more specialised, small-scale projects

•Our team of gene editing experts can offer advice and guidance at each stage of the project

•Keep up to date with the progress of your project with our dedicated online project portal.  

The platform is being expanded further through:

•Development of comprehensive profiling datasets for common cell lines to facilitate rapid engineering;

•Implementation of workflows for complex engineering – point mutations, tagging, translocations and knock out of essential genes;

•Utilising tools and technologies for screening applications – in particular coupled with in-house CRISPR arrayed and pooled library generation services.