CRISPR/Cas9-mediated genome editing is a powerful tool for the generation of genetically modified model systems in a wide variety of species. In its original function, CRISPR/Cas9 serves as an innate bacterial immune system to protect its host from viral infections. Transcription of the endogenous CRISPR locus generates a trans-activating crRNA (tracrRNA), a targeting precursor-crRNA and the Cas9 protein. The matured crRNA-tracrRNA-Cas9 complex binds to its target sites (“protospacers”), Cas9 recognizes the protospacer adjacent motif (PAM) and cleaves both strands of the target DNA to create blunt-ended dsDNA breaks. In the development of CRISPR-based engineering strategies, the system has been engineered to comprise only two components, a codon-optimised Cas9 nuclease and a chimeric guide-RNA that consists of the determinative crRNA fused to an optimised tracrRNA. In the past 3 years, CRISPR/Cas9 engineering has become the predominant genome-editing tool used to generate user-defined gene modifications in any kind of organisms.
A major problem of CRISPR/Cas9 engineering lies in the recruitment of the nuclease to genetic off-targets by undesired mismatch pairing. Although sgRNA design algorithms aim to eliminate off-target events, those are just predictions and absence thereof must be verified by experimental testing. Off-target modification in cell lines is contained, and the general accepted procedure is to generate and directly compare three or more independently established lines. When generating genetically modified animals, SNPs derived from off-target cleavage will be subsequently bred out, given they do not affect gene function, and are of a lesser concern. Targeted Sanger-sequencing of predicted off-targets is a recommended first step in model validation, and unbiased approaches (mainly GUIDE-Seq, BLESS and Digenome-Seq) are, due to their cost and time-consumption, probably restricted to CRISPR applications aiming for clinical use. Over time, the specificity of CRISPR/Cas9 engineering is continuously refined, by either using (a) DNA-Nickases (Cas9D10A) that due to inherent dual sgRNA requirement for cutting are virtually free of detectable off-target cutting (Ran et al., Cell, 2013) or by the use of (b) high-fidelity Cas9 proteins that display an dramatically increased sensitivity to mismatches in the protospacer, having an equal on-target cutting efficiency with almost no detectable off-target cleavage (Slaymaker et al., Science, 2015 and Kleinstiver et al., Nature, 2016).
General CRISPR/Cas9 Services We Offer
- We advise on the most appropriate experimental strategy to achieve a desired model system best, fast, and economic. Figure 3 gives an overview about our engineering pipeline.
- We generate verified CRISPR reagents for all CRISP/Cas9 applications: (1) validated sgRNAs (design, cloning and functional testing in Surveyor assays), (2) design custom ssODN repair templates with appropriate screening strategies, (3) design and assemble custom repair templates and large TC constructs for Cas9-assisted mESC targeting (sgRNA target inactivation, insertion of RENs for screening, suitable Southern hybridisation screens).
- We advise on all downstream screening and experimental planning events.
- We offer sgRNA in vitro transcription and purification pipeline.
- We have a repository of plasmid backbones and useful cassettes for transgenic constructs and make them available to WIMM researchers.
- We are building a verified protocol library and will help you in any aspect in your genome engineering experiment.
Service: Generation of model systems
As a general foreword (not meant as an excuse): all modifications very much depend on the cell line and the genomic environment of the locus to be targeted. DNA contained in openly accessible chromatin is much easier to target than silent or un-transcribed loci, and the underlying nucleosome architecture has an influence on cutting efficiency and HDR-events. To address those factors, we strongly recommend sgRNAs to be functionally tested by the quantitative Surveyor assay on cells, blastocysts or embryos, depending on your experimental system.
- Creation of simple KO alleles uses a single RGEN cut and relies on repair by the NHEJ pathway. Smaller insertions or deletions (“indels”) due to the imprecise manner of the NHEJ pathway will inactivate gene function in two thirds of all cutting events. Screening will be performed by simple PCR and Sanger-sequencing (de-convolution by TIDE) or in a more high throughput format using high-resolution melt analysis. This approach works well in cells and microinjected mouse oocytes.
- Generation of a defined full knock-out involves two RGENs flanking the chosen critical exon, allowing for precise excision and mending the DNA by the NHEJ pathway. The primary screen involves a simple PCR spanning the region to be deleted, and the obtained band will inform about the allelic status; selected clones will be subjected to further validation procedures. This approach works well in cells and microinjected mouse oocytes.
- Insertion of SNPs is mediated by a Cas9-cut proximal to the site of modification and by offering a 5’/3’ thioated ssODN repair donor, allowing repair by the HDR pathway. In some instances, design of asymmetric donors allows better pairing of the end-resected genomic DNA strand (Richardson et al., NBT, 2016). For positive screening, a de novo restriction site is generally co-inserted; allowing the use of a PCR based approach followed by restriction digestion. ssODN-mediated insertions work well in cell lines and to a much lesser extent in mouse oocytes.
- Insertion of larger cassettes is the most technically demanding step. Typically, 1kb homology arms each side of the cargo are required, but insertion efficiencies are generally low. Several approaches have been suggested to increase HDR rates: small-molecule inhibitors of the NHEJ pathway (Maruyama et al., Nature Biotechnology, 2015), HDR-enhancers (Song et al., Nature Communications, 2016), or specifically activating Cas9 (Gutschner et al., Cell Reports, 2016). Insertion of larger cassettes is well feasible in cell lines, however, pronuclear injection in mouse oocytes still remains inefficient. To enable facilitated generation of mouse models, our facility offers a Cas9-assisted mESC targeting pipeline based on homologous recombination. We design and assemble smaller targeting constructs containing a FRT-flanked selection marker that contain 3 to 4 inactivated sgRNA target sites (see also Figure 4). mESCs are co-transfected with a Cas9/sgRNA expression plasmid and a circular targeting construct and are selected for homologous recombination events.