CRISPR gene editing offers the potential to re-engineer biological systems and organisms. This has profound implications for basic biological research, biotherapeutic development and biotechnology. At Oxford Genetics we are keen for researchers to exploit the possibilities of CRISPR. Our CRISPR product range offers customers considerable choice and flexibility for their gene editing experiments. To accompany our product offering we have developed this general guide to CRISPR experimental design. In addition to our CRISPR product range and this guide we also offer CRISPR related services. Our gene editing and cell line development services are designed to harness the full potential of CRISPR, from optimized design of your CRISPR experiment through to complete validation of the gene editing process. Our automated approach offers a high throughput platform to a process that requires considerable optimization for reliable results. For further information view our CRISPR services page or contact us.
The CRISPR-Cas system is a prokaryotic acquired immune system in which RNA directed nucleases cleave viral DNA. The system was identified by the unusual nature of the repeating DNA sequences in bacterial genomes. These interrupted repeat DNA sequences were termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). The CRISPR-Cas system consists of CRISPR associated (Cas) nucleases and RNA sequences which will bind to the location on the genome to be cut. For more information on CRISPR view our CRISPR plasmid technologies resource.
Key components of the CRISPR-Cas system have been adapted for use as a tool for genome engineering. By replacing the endogenous RNA with the sequence of a target gene the endonuclease component (Cas) can be induced to cleave almost any DNA sequence. The prerequisite for this is the presence of a Protospacer adjacent motif (PAM) adjacent to the target region. The PAM motif is dependent upon the Cas protein selected, but for the universally employed S. pyogenes Cas9 (spCas9) the PAM is 3’NGG. These relatively simple requirements have pushed CRISPR-Cas9 to the forefront of genomic editing platforms. Yet despite it’s relative ease planning your first experiment or scaling up CRISPR genome editing can be daunting. Oxford Genetics has developed the following guide to CRISPR experimental design. As CRISPR-Cas genome editing is primarily performed in mammalian cell lines that will be the focus of the information below.
The CRISPR system is capable of both functional knockout of genes and insertion of new sequences within genes as well as other manipulations of the genome. Figure 1 illustrates some of the gene knockout or editing methods achievable with CRISPR. The first method is InDEL formation by the Non-Homologous End Joining (NHEJ) DNA repair pathway. InDELs are short insertions or deletions and require no repair template. All other methods use the Homology Directed Repair (HDR) pathway and usually require a repair template.
Figure 1. 1. InDEL formation by the non-homologous end joining (NHEJ) DNA repair pathway. InDEL formation requires no repair template. All other methods (2, 3, 4, 5) use the Homology Directed Repair (HDR) pathway and require a repair template.
Here we will provide an overview of 2 types of CRISPR experiment. Firstly, we will examine the creation of InDELs in the gene sequence. Secondly, as an example of exploiting the HDR repair pathway (which requires the presence of a repair template), we will present a guide to the insertion of a point mutation into a gene sequence.
Figure 2 InDEL Formation Workflow
Knockout by InDEL formation is the simplest experimental output to achieve using CRISPR. Transfecting cells with plasmids to express Cas9 and guide RNA sequences provides all the necessary tools. Following cleavage by Cas9, the double stranded DNA break is repaired by NHEJ. NHEJ is an error-prone process, and results in characteristic short insertions or deletions, which when targeted appropriately are often sufficient to take a gene out of frame (frameshift mutation), and thus prevent functional protein expression. Frameshift mutations may also silence gene expression by the creation of premature stop codons within the open reading frame (ORF) of the targeted gene.
Design of the guide RNA sequence is a vital step in CRISPR experiments. Your gRNA sequence must be adjacent to a PAM site (NGG for SpCas9) and be sufficiently unique compared to the rest of the genome (to reduce off-target effects). For effective knockout via InDEL it’s often necessary to design and screen multiple gRNAs against the target gene. Several publicly available bioinformatic algorithms exist for designing gRNA including one based at the Massachusetts Institute of Technology (http://crispr.mit.edu/). The MIT algorithm identifies PAM sequences within your selected target sequence and proposes potential gRNA sequences. The algorithms give each gRNA a score which is based on gRNA/DNA sequence homology and identify likelihood of cleavage at off target sites. Guide RNA sequences which are not sufficiently unique will drive Cas9 to offsite targets leading to non-target DNA cleavage. Evidence from our scientists suggests that off target cleavage may be more common than certain algorithms predict and therefore we recommend that assessing this should be factored into the experimental design, should this be important to the downstream application.
Oxford Genetics offers two types of Cas9 expressing plasmids for researchers to perform gene knock out by InDEL formation. The bifunctional plasmid method uses one plasmid to deliver 1-2 gRNAs and expresses Cas9. The two-plasmid method uses a single plasmid to deliver 1-2 gRNAs and a second plasmid for Cas9 expression. It is widely noted that the expression level of the cleavage nuclease Cas9 influences the specificity of the CRISPR/Cas system. To find the most appropriate expression level of Cas9 for your experimental system, Oxford Genetics offers a variety of plasmids each with a different promoter driving Cas9 expression (Table 1).
|Cas9 Expressing Plasmids||OG5202||OG5205||OG5208||OG5210||OG3570||OG3573||OG5214||OG5217||OG5220||OG4155||OG3579|
Table 1 Cas9 expressing plasmids listed according to the promoter which drives the expression of Cas9.
Depending on your preferred cell line or cell type, transfection conditions may need to optimized experimentally. There are multiple different options for plasmid delivery - chemical transfection such as lipid and calcium phosphate methods are generally applicable for immortalized cell lines whereas, electroporation is suitable for delivery of your Cas9 plasmid and gRNA to both immortalized cell lines, isolated stem cells and primary cultured cells.
If you have a fluorescent protein marker on your plasmid, you can use FACS to enrich the cells that received Cas9 and your gRNA. Similarly, if your plasmid conveys resistance to an antibiotic this can also be used to select for cells that received Cas9 and your gRNA. For post transfection selection purposes Oxford Genetics has designed plasmids which express either the fluorescent marker EGFP or a gene which conveys resistance to puromycin.
|Cas9 Expressing Plasmids||OG3565||OG5210||OG3576||OG3557||OG5206||OG4761||OG4764|
Table 2 Cas9 expressing plasmids listed according to post transfection selection marker
After transfection there are several methods available to confirm the efficiency of your CRISPR experiment. When using a single gRNA, the size change resulting from the InDEL is insufficient to detect by PCR and gel electrophoresis. Therefore two alternative methods are standardly employed – a surveyor/T7EI assay, or a TIDE assay.
Surveyor/T7EI Assay-The basis of this assay is the ability of the surveyor nuclease to detect SNPs between alleles and cleave the double stranded DNA at the 3′ side. To conduct this assay, you must extract your DNA, amplify your target region, denature and rehybridize the DNA, allowing the mutant and wild type strands to anneal. Following this treat the samples with the surveyor nuclease. Analysis of the DNA on agarose gel will show cleaved fragments indicating the presence on InDELs.
TIDE Assay-The TIDE assay involves Sanger sequencing of the PCR product and inferring Cas9 cutting efficiency by deconvoluting the mixed spectral peaks obtained during the sequencing run. Alternatively, when more than 1 gRNA is used per target gene, standard PCR and gel electrophoresis may be used, allowing for the anticipated size differential of the resultant products. With all CRISPR experiments, NGS analysis provides the gold standard in genotyping, and is used at Oxford Genetics routinely in our editing workflows.
Finally, depending on your downstream application it is often important to verify changes at the protein level, normally using a western blot assay.
Overview - Introduction of Point Mutations
Figure 3 Introduction of Point Mutation Workflow
As mentioned previously it is possible to do more than simply knockout genes using CRISPR technologies. By exploiting the more precise HDR pathway it is possible to make a specific edit of the genomic DNA to induce a mutation such as a single nucleotide polymorphism (SNP). This requires a DNA repair template that has arms of homology that flank the region of DNA where the cut is designed to occur. The length of the repair template depends on the application, and how large a modification is being made. It can be a single stranded oligonucleotide, double stranded oligonucleotide or a double stranded donor plasmid. Typically, SNPs or small modification would utilise single stranded templates, and larger modifications, incorporating fluorescent or selectable markers, would use double stranded plasmid or linear templates. While the repair template must contain the desired mutation it must not, however, contain the PAM site used by the gRNA, this is to prevent the repair template itself becoming a target for Cas9. Due to the low efficiency of HDR compared to NHEJ, generally only small percentage of cells may contain the desired mutation. Therefore, having an efficient screen at the pool and clonal level is highly important.
The repair template must contain:
Figure 4 Insertion of a synthetic construct by homologous recombination. Cas9 cleaves the genomic DNA (blue) at the target site leading to a double strand break. If a repair template is supplied the break can be repaired by homologous recombination. In this example the repair template is being used to knock-in an expression cassette. The cassette is flanked by 800 bp of DNA sequence with homology to the genome on either side of the target site. The actual sites of recombination (red crosses) may vary depending on the position of the Holiday junctions when they are resolved.
Figure 5 Arms of homology. Note that the repair template does not contain the complete gRNA target sequence. Each arm of homology begins 1 bp away from the likely break site. The length of the arms of homology typically range from 80bp for single stranded oligos, to 200-800bp for double stranded templates. It is also important that the repair template does not contain the complete gRNA target sequence otherwise the donor vector may be cleaved by Cas9. This can be done by placing the insert in the middle of the target sequence (as in figure 5) or by altering the PAM sequence in the repair template. If the PAM site is within a coding region, care must be taken to make sure the alteration is a silent mutation.
As mentioned in the InDEL overview above, select a plasmid which is appropriate for your experimental system. View our catalogue of CRISPR plasmids or contact us for bespoke Cas9 plasmid design and synthesis.
CRISPR mediated introduction of a sequence into a gene requires promoting the HDR DNA repair pathway over the more commonly used NHEJ. Since HDR occurs during the S and G2 phases of the cell cycle many researchers try to enhance HDR by synchronizing the cells in these phases of the cell cycle. Alternative strategies include chemically, or genetically inhibiting genes involved in NHEJ. One such molecule is SCR7 which promotes the HDR pathway by inhibition of DNA ligase IV.
4. Post Transfection Enrichment / Conformation of Point Mutation Introduction
The result of the point mutation introduction will be a mixed population of cells. Some cells will not have been edited, others will have one allele edited, and others will have both alleles edited. The first step is to enrich the cells which contain the inserted sequence. If the inserted sequence contained a selection marker (puromycin resistance gene/ EGFP) this can be used to sort the population. However, not all of this population will have the sequence inserted in the proper orientation. Following this enrichment, we recommend PCR with primers based at the homology arm junctions to ensure the sequence has been inserted completely and at the correct point. Additional PCR with primers located at internal sites of the inserted sequence will ensure the sequence has maintained correct orientation.
The basic gene editing made possible by CRISPR is an elegantly simple biological process. However, producing highly specific gene edits can be challenging. It requires a scale of production at several steps including gRNA design and synthesis, optimization factors such Cas9 expression and post transfection cell selection. At Oxford Genetics we are uniquely placed to meet these challenges. Our substantial experience in DNA engineering combined with a highly automated approach to production gives us both the knowledge and scale to meet even highly complex gene editing needs.
CRISPR Pooled and Arrayed Libraries
Oxford Genetics’ custom CRISPR-Cas9 libraries are available in either a pooled or an arrayed format. Each plasmid in the libraries contain a Cas9 and a guide RNA (gRNA) toward a different target gene, supplied in either a plasmid or lentiviral particle format. These can then be delivered into cells enabling rapid, effective knockout of target genes, and making them suitable for typical screening work flows. These screens can be targeted against a bespoke selection of gene or against components of signalling pathway such as the kinome.
As part of our CRISPR service offering Oxford Genetics offers methods of CRISPR delivery other than plasmid transfection. These methods include inducible and stable genomic expression of Cas9 as well as exogenous introduction of Cas9. Optimised delivery of Cas9 can significantly decrease off target effects increasing the quality of your results.
CRISPR Experimental Design
Our scientists will collaborate with you to design an optimized experiment plan depending on your requirements. We have extensive options in terms of gRNA design, delivery methods, type of genome edit, population selection and edit confirmation procedures.
Proprietary gRNA design ensures the production of gRNA with the highest level of specificity and activity to guide Cas9 to the correct site of the target gene and ensure cleavage by Cas9.
Post Transfection Enrichment/Confirmation of Edit
As part of our service offering Oxford Genetics offering several methods for enrichment of your edited population. These include among others fluorescent cell sorting and introduction of antibiotic resistance. These selection markers come in several different formats and can be customized to your experimental needs. We also offer PCR and NGS based methods for confirming the correct edit of the genome has been made. The optimal method to validate your gene edit can be selected and performed in house by our experienced scientific team.