Services and Facilities
CRISPR/Cas9-Directed Gene Editing
The CRISPR, Vector and Transgenic Mouse Core has developed and is pleased to offer Cas9 nuclease-mediated approaches for CRISPR genome editing in human iPSC lines, primary cells, mouse embryos, and mouse embryonic stem (ES) cells. Cas9 can be provided by a plasmid or viral vector that encodes Cas9, or as Cas9 protein. For genome editing, the Core frequently uses Cas9 ribonucleoprotein (RNP) consisting of the Cas9 protein in complex with the targeting gRNA. PAM sites in the repair oligos are mutated as needed. For knock-in of genes, a “safe harbor” locus is often used, such as the ROSA26 or AAVS1 loci. Safe harbor sites are sites in the genome at which genes can be inserted without disrupting the expression of adjacent or more distant endogenous genes.
For CRISPR genome editing in mice, the Core uses electroporation for deletions and small edits (such as point mutations). We have optimized the electroporation method, using Cas9 RNP, so that the efficiency is close to 100%. An advantage is that many more mouse embryos in a pool can be targeted by electroporation than by individual microinjection, which greatly increases speed and editing efficiency. Moreover, electroporation is effective in virtually any mouse strain, thus facilitating the gene editing in mice of complex genetic background.
For more complex genome editing, such as inserting two LoxP sites flanking an exon on the same DNA strand, or inserting larger fragments, electroporation is less effective. The Core therefore uses CRISPR editing of ES cells, similar to the strategy used for generating cell lines. Alternatively, one LoxP site can be inserted by electroporation, the mouse colony can be expanded, and embryos from mice homozygous for the edit can then be used for electroporation to insert the second LoxP site. This strategy is very efficient, but takes longer than the ES cell approach because of the required mouse breeding to generate donors for insertion of the second LoxP site. The timeline from initial design to delivery of custom-targeting and associated expression plasmids for cell transfection is 2-3 weeks. The Core also provides advice and services related to CRISPR/Cas9 technology, including design, screening, genotyping, and sequencing of modified cell clones.
Other CRISPR Applications
CRISPR is being increasingly used for applications other than genome editing, and at the request of DRC affiliates, the Core offers such CRISPR services. The most frequently requested are CRISPR methods for transcriptional activation and repression, and CRISPR libraries for screening.
Adenovirus Associated Vectors (AAV) for Use in Animals, Tissues and Cells
DNA virus vectors based on AAV have the advantage that very high titers can be produced and a wide range of capsid serotypes can be utilized to facilitate transduction of target tissues in animals. While the packaging capacity of AAV is limited to ~4.7 kb, this size accommodates most transgene cassettes. The viral genomes form stable high molecular weight concatemers that persist for years in non-dividing tissues making them both safe and effective transduction tools. AAV vectors transduce non-dividing cells effectively and rarely integrate. The Core’s vector production methods use a plasmid co-transfection system, which prevents production of wildtype AAV, hence they can be used under BSL-1 containment.
The CRISPR, Vector and Transgenic Mouse Core produces AAV vectors designed to match the needs of our users. This includes a choice of ubiquitous, tissue-specific or user-supplied gene regulatory elements (‘promoter’) driving a transgene of interest. Our Core has several pre-made plasmids carrying AAV2 inverted terminal repeats (ITRs), several promoter elements, reporter genes and a strong PolyA site. These plasmids are customized for the use of each individual investigator by standard cloning methods, and can then be pseudotyped with capsid proteins from a large set of AAV capsid genes to help tailor transduction profiles of the vector. The Core currently has helper plasmids carrying capsid genes from numerous serotypes including AAV 1-9, rh74, 2i8, AAVMyo, and several DJ variants. Typical preps are done by growing cells in 1.5 L roller bottles in a 96 bottle roller rack incubators, which can grow up to 192 bottles at once. However, many users require small scale preps which are produced in 15 cm dishes. Our current production methods are similar for all serotypes, with differences in the final purification steps depending on the capsid used for pseudotyping. In general, we use a 2 plasmid co-transfection system followed by harvesting of both the cell supernatant and pellets to increase yield. Heparin-binding serotypes are purified by affinity chromatography, sucrose gradients and, depending on needs, on CsCl gradients. Most others are purified by a conventional purification method utilizing several ultracentrifugation steps and a chromatographic purification protocol utilizing cationic exchange columns. Purified vectors are titered by both Southern analysis and qPCR.
Recombinant adeno-associated viruses are important tools for gene delivery and expression. AAV has not been reported to cause any diseases. Together with its replication defective nature, AAV has a good safety profile to be used in gene transfer in vivo, and as potential gene therapy vehicles. Recombinant AAV is capable of infecting a broad range of cell types including non-dividing cells and remaining as concatemers for long-term expression. Compared with other viral vectors such as adenovirus, AAV elicits very mild immune response in animal models.
Lentiviral Vectors for Use in Animals, Tissues and Cells
The CRISPR, Vector and Transgenic Mouse Core provides well characterized third generation VSV-G pseudotyped lentivirus self-inactivating (SIN) vectors for use in cells, tissues, or intact animals. The lentivirus expression plasmid, pRRL-cPPT-CMV-X-PRE-SIN, incorporates a central polypurine tract (cPPT) and a posttranscriptional regulatory element from human hepatitis B virus (PRE). Expression plasmids encoding an enhanced GFP (eGFP) reporter, luciferase, or β-galactosidase with and without a nuclear localization signal serve as positive controls and quality controls. The Core has pioneered the use of IRES to provide coordinate expression of two or more genes of interest in bicistronic retrovirus vectors and has constructed three bicistronic vectors encoding encephalomyocarditis virus (EMCV) IRES, a multiple cloning site and a marker gene, enhanced GFP (pRRL-cPPT-CMV-X-EMCV-eGFP-PRE-sin). To achieve the goal of coordinate gene expression at equivalent and high levels the Core employs constructs encoding peptide 2A. These constructs enable affiliate investigators to monitor the expression of both their gene of interest and a marker gene. This is a major benefit where the investigational gene is not easily monitored and also permits determination of virus titer.
The Core also generates lentiviruses encoding promoters to achieve cell and tissue specific transgene expression for use in vitro and in vivo. For example, the Core has generated vectors encoding a hepatocyte specific promoter, an adipocyte-specific adiponectin promoter, and muscle actin or MCK promoters. Furthermore, the Core generates lentiviruses encoding small hairpin RNA (shRNA) to provide sustained gene silencing in vitro and in vivo. All lentiviruses are titered before they are given to affiliate investigators. Typical Lv titers are in the range of 1e9 particles per ml from 10×10 cm plates.
Replication Competent Retrovirus (RCR) Assay
Lentiviral vector preparations produced by the Vector and Transgenic Mouse Core can be tested using the RCR assay before use if the investigator prefers to use them at BSL1 safety levels (non-RCR tested vectors can be used at BSL2). The Core will direct investigators to appropriate sites for this service. The Core has not yet found replication competent virus in any lentivirus preparations, in agreement with reports in the literature.
Development of Genotyping Methods and Sequencing to Validate Genome Editing in Mice and Cells, and Specialized Molecular Biology Methods Not Routinely Performed in Affiliates’ Laboratories
The CRISPR, Vector and Transgenic Mouse Core develops genotyping methods for all genome edited cell lines and mice. Genotyping can be performed in the Core to select cell clones and mice with correct edits for experiments and colony expansion. Alternatively, the protocols are provided to the affiliate investigator for use in their own laboratories. The Core performs sequencing of all vectors to validate correct sequences.
The Core offers molecular biology services not routinely performed in DRC affiliates’ labs. These include real-time PCR with mRNA copy number quantification (using an Agilent/Stratagene Mx3005P PCR instrument), cloning and targeting vector design and construction, and site-directed mutagenesis. The Core offers gene synthesis either in-house or using the local company Blue Heron Biotechnology. This option can be especially useful in accurately obtaining long genes, vectors, or complex constructs, when no native sequences are available or when rare RNA transcripts are required. Synthesized genes are delivered as 3-5 μg of purified plasmid DNA with the bacterial strain containing the plasmid and sequencing/trace data.
Cost-effective Cryopreservation through the UW Transgenic Resources Program
The CRISPR, Vector and Transgenic Mouse Core offsets the cost for Diabetes Research Center affiliate investigators to use the Transgenic Resources Program for cryopreservation.
Consultation and Training
An important aspect of the CRISPR, Vector and Transgenic Mouse Core’s function is to offer consultation concerning virus production and use, specific viruses that best satisfy each affiliate investigator’s needs, CRISPR/Cas9 methods, generation of genetically engineered mice, and molecular biology techniques, e.g. qRT-PCR with primer/probe design. These services are provided by the core directors. In addition, the Core offers training in any technique that is carried out by the Vector and Transgenic Mouse Core.
The CRISPR, Vector and Transgenic Mouse Core’s laboratories are located in the F building and S building at the University of Washington’s South Lake Union Campus. The Vector and Transgenic Mouse Core space consists of wet lab space, BSL2+ tissue culture rooms, and procedure rooms.
Key equipment within the Vector and Transgenic Mouse Core includes PCR machines (ABI), a Nanodrop (Thermo), Mx3005P and Mx4000 real-time PCR systems (Aglilent) and equipment for virus production and purification. The Vector and Transgenic Mouse Core has access to shared equipment in the UW Medicine Diabetes Institute, including ABI real-time PCR machines, MaxWell RNA/DNA isolators, fluorescence and luminescence plate readers, gel scanners, and flow cytometers and cell sorters.