CRISPR/Cas9 pipeline

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

 

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Figure 3: Overview of our CRISPR/Cas9 engineering pipeline. (A) We start in determining the best available sgRNAs for your needs, trying to minimise potential genic off-target effects. (B) We clone sgRNAs into the vector that is most appropriate for your needs. (C) We do a functional validation in cells using the Surveyor assay in orthologous cell lines or embryo systems. (D) We offer help in the determination of how to best deliver Cas9 and the sgRNAs into your model system. We are able to mediate contact to expert groups within the WIMM and within Oxford University. (E) We help you design appropriate screening strategies to assess target cleavage and (F) help you to assess the frequency of successful NHEJ or HDR events.

 

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.

 

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Figure 4: CRISPR/Cas9 assisted mESC targeting. Overview of a prototypical mini targeting construct. The plasmid remains supercoiled while Cas9 generates the DSB in the target genomic DNA, reducing potential random insertion events of the donor cassette. Homology arms are rather short (1 to 2kb) and thus allow for initial PCR screening.