Increasingly the literature suggests that CRISPR's potential for off-target effects is not as “bad” as originally thought. Here are a few things to set your mind at ease if you’re working with the CRISPR-Cas9 system:
Early on in our CRISPR use at Horizon, we learned that that when it comes to guide RNA design not all sgRNAs are created equal. In fact what makes a good guide is still the topic of a great deal of research in the field – and aside of the consensus that a guide with a guanine at position -1 is more likely to have a higher activity, we are still a way from being 100% confident in predicting in silico guide activity in vitro/vivo.
The CRISPR/Cas9 system has been rapidly adapted to practically every model system for its ease to generate and high efficiencies to cleave target DNA. But unlike our experience with Zinc Finger Nucleases, in the human, rat and mouse cell lines we tried successful co-transfection of Cas9 mRNA and sgRNA was cell-line dependent, and often resulted in either very low or no cleavage activities.
However, sequential transfection of cells with Cas9 DNA first, and sgRNA followed 24 hrs later, reliably produced good level of activity, indicating the requirement of Cas9 presence at the time of introduction of sgRNA. Not surprisingly, creation of a cell line stably expressing Cas9 led to consistently high cleavage activities upon transfection of sgRNAs. Transfection of recombinant Cas9 protein pre-complexed with sgRNA (ribonucleoprotein particles, or RNPs) led to efficient cleavage as well.
On the other hand, when Cas9 mRNA and sgRNAs are co-microinjected into single cell embryos, it produces target cleavage as efficiently as RNPs to produce straight KOs and large deletions between two target sites, again raising a question of local concentrations of Cas9 protein and sgRNA.
Below we summarize some of the work we've done optimizing delivery of CRISPR-Cas9, which which can be read in full publication form in Human Gene Therapy here.
Here you'll find a complete list of our most frequently asked questions relating to X-MAN cell lines. How are they made, how are they validated, and how can you use them? Read on to find out.
Whether it's a stock you've expanded following it's arrival from the cell bank, or a clone you've carefully nurtured from the single cell, banking down your cells in the right way is crucial if you're going to be able to return to them time and again, and revive them quickly so that you can get on with your experiments.
Here's our protocol for banking human cell lines:
Transfection or electroporation is used to efficiently introduce the nucleic acids required for CRISPR cell line engineering into a cell line. The type and number of nucleic acids (usually plasmids) being introduced into a cell line will depend on the engineering event being undertaken. At its most simple, gene knockouts can be achieved by transfection of a plasmid expressing wild-type Cas9 along with a guide RNA (gRNA) to the gene that is being knocked out.
If you've followed our guide to planning a successful genome editing experiment, then you'll hopefully be working in optimal conditions, and have a good idea of your guide's editing efficiency. This number should give you a rough idea of how many clones you're going to have to screen to find a targeted clones. Keep in mind the following:
Once a clone has been identified positive for targeting in the initial screen, the cells should be expanded to a 48-well plate. Expansion of the cells can usually occur at 1-4 days post screen.
We have combined our proprietary genome editing technology with large scale RNAi screening capabilities to allow identification of novel drug targets.
Isogenic cell lines provide genetically defined, patient-relevant, predictive in vitro models of genetic disease. We are further extending their application within targeted drug discovery with the development of new reporter disease models using our gene engineering technology. These models combine Horizon's cell lines with the endogenous gene reporting capabilities in the form of NanoLuc® luciferase and HaloTag® reporter technologies.
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.
Reporter gene assays are widely used to study the regulation of gene expression. We have developed a suite of endogenous reporter cell lines which measure natural levels of protein expression and promoter activity. By measuring at the endogenous level, this system provides an advantage over other technologies which use exogenous plasmid-based overexpression systems.
The term synthetic lethality was first used in 1922 by C.Bridges1 to describe events where two non-lethal mutations are brought together in combination to cause cell death. Synthetic lethality offers significant potential in the field of cancer research, with many groups focusing on the selective inhibition of target proteins that are lethal to only those cells harboring a mutated cancer gene.
At Horizon Discovery we process tens of thousands of microtiter plates per year in support of our cell based screening operation and its library of thousands of commercially available and proprietary cell lines.
There exist now a range of techniques to perform genome editing, such as ZFN, CRISPR, TALENS and AAV, each with their own strengths and weaknesses. However, one consistent element that has a significant impact on the success of that editing event when generating an isogenic cell line is the choice of parental cell line to be engineered.
Recombinant adeno associated virus (rAAV) is a precise and effective method to introduce defined changes into endogenous genes and rAAV vectors can stimulate homologous recombination (HR) up to 1000-fold over that seen using plasmids.
Nuclease based approaches like CRISPR-Cas9, ZFNs and TALENs facilitate targeted modification of genomes by inducing double-strand breaks (DSBs) within chromosomes at specified locations. This stimulates the natural DNA-repair mechanisms of homologous recombination and non-homologous end joining.
Plasmid DNA, PCR products, and single stranded oligonucleotides are routinely used as donors to introduce specific changes at the DSB site. The efficiency of introducing a desired change is dependent on many factors including
- the type of donor
- the length of homology
- the complexity of the desired change
- characteristics specific to the cell line
We have looked at whether rAAV vectors can be used as donors for DNA modification to obtain higher efficiencies than seen with other donor approaches.
KRAS is one of the most frequently mutated genes in cancer, but targeting KRAS with potent small molecules has proved to be difficult. Moreover, although inhibitors of BRAF and MEK, which are downstream targets of KRAS, have been developed, they have transient benefits only in patients with melanoma who have mutated BRAF. Therefore, an effective therapy for KRAS-driven tumours remains a pressing unmet medical need.
A number of scientists and clinicians in Glasgow are currently participating in the National Lung Matrix Trial (NLMT), which aims to expand the use of stratified medicine approaches for the treatment of non-small cell lung carcinoma (NSCLC), by testing the efficiency of selected biomarker-targeted therapy combinations.
Neomorphic mutations targeting amino acid R132 of the TCA cycle enzyme, IDH1, have been identified in multiple cancer types and lead to a build up of (R)-2-hydroxyglutarate (R)-2HG.
Several mechanisms have been proposed to account for mutant-IDH1-mediated transformation:
- (R)-2HG may compete with alpha-ketoglutarate (α-KG) dependent enzymes that act as tumour suppressors such as TET2 or EGLN
- (R)-2HG might inhibit electron transport chain function
- Rapid (R)-2HG generation may deplete the cellular pool of α-KG leading to depletion of NADPH.
According to some reports in the literature, heterodimer formation between mutant and wild-type alleles of IDH1 is important for the production of high levels of (R)-2HG1.
We were interested in exploring novel ways to target tumour cells bearing mutant IDH1 alleles that were distinct from the obvious opportunity available to identify mutant-specific IDH1 inhibitors. The potential metabolic vulnerabilities of mutant IDH1 cancers raised the possibility that wild-type IDH1 might be essential for tumourigenesis or tumour maintenance in this context.
We therefore employed Horizon’s rAAV-mediated homologous recombination gene engineering technology to generate conditional knockouts of the IDH1+ or IDH1R132C alleles in the fibrosarcoma cell line, HT1080.
The discovery of the CRISPR‐Cas system in bacteria has initiated an impressive array of innovations that have enabled the use of the RNA‐guided Cas9 nuclease in functional genomic screens. At Horizon, we have embraced these developments, as they provide new opportunities for drug target identification and validation. The case studies presented in this below highlight how we use this technology to successfully conduct genome wide and focused sgRNA library screens and to verify whether specific genes are required for the survival and/or proliferation of cancer cell lines.
A number of siRNA and shRNA screens have identified targets that exhibit differential dependencies between KRAS mutant and KRAS wild-type tumours, but there is poor overlap between these published studies. Next generation screens that exploit both isogenic cell lines and cancer cell panels, and use a combination of knockdown (si/shRNA) and knock-out (CRISPR-Cas9-sgRNA) methodologies might be more effective at identifying novel targets that withstand validation. However, if we are to detect co-dependence as well as synthetic lethal interactions, screens must be performed under conditions where mutant KRAS alleles are essential for growth.
The emergence of RAS mutations is a key mechanism of acquired resistance to MAPK-pathway targeted agents in a number of cancers. The preclinical evaluation of targeted agents traditionally relies on panels of genetically unrelated cell lines grown as 2D monocultures. The heterogeneous nature of these panels makes identifying genotype-specific responses a challenge. In addition, 2D assays do not accurately mimic the tumour microenvironment and so add to the difficulty in interpreting which cellular responses to targeted agents will have relevance in vivo.
Ras mutations are amongst the most commonly occurring mutations in human cancer, present in approximately 49% of colorectal and 20% of lung cancers. Of these, mutations in K-Ras G12 and G13 are the most common. Understanding the role of mutant K-Ras in modulating drug response is critical to the successful development of novel therapeutics, and has been hampered by the lack of suitable in vitro tools.
Laboratory-based CRISPR gene editing has only been around a short while but is already revolutionizing the way we do biological research - allowing scientists to study gene function in more robust or even previously un-imagined ways.
Much information about the role of specific genes in fundamental biological processes and the onset and progression of genetic disease has been gleaned by researchers having the ability to selectively alter the genomic composition of individual genes and study the consequences. This approach enables researchers to observe the effects of a specific mutation, SNP or deletion in combination with the added layers of regulation present within the cell, including post-translational modification, epigenetic changes associated with chromatin structure, and transcriptional mechanisms.
While the Nobel prize winning work of Capecchi, Evans and, and Smithies introduced the concept of manipulating the genome of mouse ES cells, the ability to manipulate a broader range of cell types, human cells in particular, remained a significant challenge for some time. The more recent discoveries of nuclease based targeting technologies like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) has greatly increased interest in genome editing and provided even more efficient platforms for achieving targeted genome modification. Despite the increases in efficiencies these technologies offer, there are still a wide range of factors that influence success and failure in genome editing.
While the scope of genome editing is very broad and includes whole organisms, this article will focus on the issues faced primarily by scientists attempting to modify the genome of immortalized cell lines. The ability to create isogenic cell lines in which the genome editing event is the sole differentiator between the phenotypes of two cells is a powerful tool. Despite all the recent developments and improvements in targeting platforms, certain challenges remain and the role played by the choice of cell line in order to achieve success cannot be understated.
Thanks to next generation sequencing (NGS), we are starting to understand the mutational changes that occur across the board in the cancer genome. With this knowledge comes potential – novel mutated genes and the proteins that they encode are candidates for prognostic markers and/or new drug targets.
It’s hard to keep up with the rapidly expanding world of CRISPR, and it’s starting to feel like CRISPR screens are being published every week, taking the technique from the cutting edge to the mainstream.
Due to their larger size and higher intelligence rats are superior to mice for many neuroscience fields, but for historical reasons including challenges with genome editing they have often been overlooked in preference to the mouse. However, programmable nuclease technologies such as zinc finger nucleases (ZFNs) and CRISPR/Cas9 system have made engineering rat models much easier, and in this manner it is now possible to introduce the Cre-LoxP system into the rat.
Using gene editing we have created a suite of rat models for optogenetics research, consisting of various neuron-specific Cre drivers, Cre activity-dependent fluorescent reporter and opsin-expression rat lines. You can find our complete characterization of these rats in our recent publication in PLOS ONE, but below we detail some of the analysis that demonstrates that the Cre expression in these Cre driver rats faithfully recapitulates that of the respective endogenous gene.
Following transfection of CRISPR reagents, cells will need to be single cell diluted to obtain a clonal population. There are several ways of doing this. Below we detail the single cell dilution protocol used at Horizon.
If a cell line tolerates being single cell diluted then plating 96-well plates at 1 cell per well in standard cell culture media is appropriate.
If the cell line does not tolerate single cell dilution in standard media, then the use of conditioned media can often improve clone recovery. If the use of conditioned media in 96 well plates does not improve the recovery of clones, cells can be plated to large tissue culture dishes and individual colonies picked.
Cell lines remain one of the most important research tools in many labs today, be it for the study of basic subcellular processes or disease biology.
The cell line selection is therefore a critical first step in any research project. Here’s a number of factors to consider, as well as resources to refer to when choosing your cell line (or lines).
Given the time intensive nature of gene engineering, the relatively straightforward and quick (<1 week) process of guide RNA validation can save weeks of cell culture and hours of bench time and ensure you're work with the most optimized sgRNA available. Further to this, a clear idea of gRNA activity will provide insights into how many clones need to be screened to identify a positive.