Module 2: Gene Editing and Virus Production

Modulation of gene expression and gene editing in biological systems represent essential tools to interrogate the mechanisms and pathogenesis of human diseases. The Gene Editing and Virus Production (GEVP) Module provides full-service DNA production, gene editing constructs, and validation, as well as lentivirus and AAV production/purification to vision scientists at UTSW and neighboring institutions. This advanced production facility includes not only routine workhorse instruments (PCR thermal cyclers, DNA imaging platforms), but high-end instrumentation including an ultracentrifuge, quantitative PCR (QuantStudio6), and sensitive western blotting (LI-COR) as well. Dr. Park and his staff are experts in all aspects of this Module as evidenced by multiple publications using each technique. This Module is a unique resource that will allow vision scientists to test new hypotheses, explore biology, and free themselves from labor- and infrastructure-intensive procedures.

The goals of this Module are to:

1) Disseminate leading-edge gene editing tools (e.g. versatile versions of Cas9, base and prime editing, as well as endonuclease deficient Cas9 for epigenetic modulation).
2) Enable investigators to develop and test unique DNA and RNA constructs in their research.
3) Free investigators from processing, maintenance, and record keeping related to plasmid DNA preparation and storage
4) Provide high quality, high titer lentivirus and AAV for generation of stable cells and in vivo work.
5) Confirm proper expression and localization of generated mammalian constructs prior to disseminating them to investigators.
6) Provide custom validation of shRNA and CRISPR-mediated gene expression, overexpression, and knockdown.

Facilities and Resources

The GEVP Module laboratories are on the 7th floor of the Florence (E) Building in close proximity to the laboratories in the Department of Ophthalmology. The Facility is housed in five rooms (all within a two-minute walk of each other, each dedicated to the specific Core function) for a total of 1610 ft² of space. The GEVP Module contains resources for DNA synthesis, mutagenesis, purification, sterile cell culture, ultracentrifugation, and transcriptional/proteomic evaluation. 

In addition to the services provided directly by the GEVP Module described above, this Core will utilize, or facilitate the utilization of established UTSW Core Services. All sequencing will be performed by the UTSW McDermott Sanger Sequencing Core that provides next business day results (drop-off box located in adjacent L building, picked up multiple times a day), and appropriate AAV vectors (along with required replication/capsid plasmids) will be generated and large-scale purified for eventual packaging by the UTSW AAV Core, located on the UT Southwestern North Campus, NA2.500 (UTSW Viral Vector Facility, directed by Dr. Steven Gray).

Equipment

• Molecular biology laboratories (E7.132 and E7.239; 500 ft²⁾

  These two labs have the following equipment that is relevant to the day-to-day function of this Module: ACD HybEZ™ II Hybridization System for RNAscope and BaseScope, a clean bench, four PCR machines (2 of which are capable of gradient PCRs), 4 independent gel rigs and power supplies, a LI-COR Fc for imaging DNA gels, a Safe Imager for excising DNA bands, two shaker incubators, three dry baths of varying temperature, one water bath, one refrigerated thermal mixer, one refrigerator, one -20°C freezer.

• Viral production laboratories (E7.130, E7.238; 960 ft²)

  These two labs have the following equipment that is relevant to the day-to-day function of this Module: two BSL2 biosafety cabinets, four cell culture incubators, one Akta go, one Heracell cell culture shaking incubator, a Sorvall floor model centrifuge, a Sorvall ultracentrifuge, a refrigerated minicentrifuge, two tabletop centrifuges (one refrigerated), one refrigerator, one -80oC freezer.

• Additional supporting instrumentation: (E7.239, 150 ft² in E7.212)

  Nanodrop for DNA/RNA/protein quantification (located in the common equipment lab, E7.212). LI-COR CLx for imaging western blotting after transient transfection (located in E7.239). QuantStudio6 to confirm expression levels by qPCR after transient transfection (located in E7.212). Additional -80° freezer space (E7.212) for backup storage of single use viral aliquots. An Attune NxT Flow Cytometer from Life Technologies and a Sony SH800 Cell Sorter are available for use as needed in the Stem Cell, Organoid, and Cell Phenotyping Module.

Services and Methodologies

Recombinant DNA synthesis has been a routine laboratory task for more than 25 years and is a critical foundation for exploring new ideas on how to manipulate and/or restore biology. It is understood that even single base pair alterations (synonymous or mutations) can alter the encoded protein. Thus, it is critical to maintain pristine records to ensure DNA fidelity and rigor/reproducibility of subsequent experiments. Yet, in practice, this can be a painstaking and laborious task requiring dedicated personnel to ensure consistency and maintain standards. The GEVP Module will enable users to generate virtually any construct for their studies. These constructs will then be used for either overexpression or knockdown/out studies. DNA constructs will be fully sequenced, and stocks (glycerol and plasmid) will be well documented and maintained in Microsoft Access spreadsheets and SnapGene files. Additional contributions that this Module will make to ensure rigor and reproducibility include validation of the produced DNA constructs at the transcript level using highly specific TaqMan probes and at the protein level (western blot and immunocytochemistry) using knockout/down validated antibodies. Additional knockout/down validation at the tissue level will be made available using RNAscope and Basescope probes (Advanced Cell Diagnostics). Furthermore, centralizing, and confining virus production into a single facility minimizes expenses of maintaining sterile BSL2 facilities and maximizes containment of infectious biohazard material.

• Generation of new DNA constructs and shRNAs

  For projects to test expression of a gene of interest in any given system, we will first check the UTSW Ophthalmology DNA repository which contains ~850 unique DNA constructs or open sources (like Addgene and DNASU), which will provide access to over 250,000 unique plasmids in a variety of species. If cDNA cannot be obtained from the above sources, we will generate the cDNA using PCR amplification from target tissue mRNA. The cDNA of interest will be cloned (ligated) into entry vectors (e.g. pENTR1A Gateway Entry Vector) using appropriate restriction sites. All DNA clones will be verified fully by sequencing (including the promoter and 3’ UTR, if applicable, McDermott Sanger Sequencing Core). Using the entry vector, we will introduce appropriate tags (such as HA, FLAG, myc) using Q5 mutagenesis (NEB) and appropriate NEBulider-designed primers (NEB). For insertions up to 30 nucleotides, or any size deletion, the Q5 mutagenesis kit will also be used. For larger insertions and multiple gene assemblies into one construct, Gibson Assembly (NEB HiFi Master Mix) will be used. If a construct cannot be appropriately synthesized by these means, we will purchase a gBlock oligonucleotide of the sequence, ligate it into pENTR1A, and screen the resulting clones to identify the correct sequence. Each correct pENTR1A clone will be stored as a glycerol stock for safekeeping and a SnapGene file will be generated for each investigator (each investigator will also be given a software license of the full version of SnapGene to allow for viewing and manipulation of the sequence).

  The pENTR1A constructs will be shuttled into one of various vectors depending on ultimate need: An alternative pCAG (chicken ß actin/CMV hybrid promoter) vector will be used as a destination vector if we find silencing of the CMV promoter in the above vectors based on subsequent follow up experiments in mammalian cells. Additionally, if desired, the constructs can be ligated into adeno-associated virus (AAV) vectors for ultimate production.

  For long-term knockdown, shRNA is a widely used method to specifically reduce transcript levels. We will purchase and test a series of validated, commercially available shRNA clones against transcripts of interest (Sigma) in lentivirus vector backbones driven by either the U6 or H1 promoter. Alternatively, we can select optimal shRNAs sequences using online tools, including InvivoGen siRNA Wizard. These constructs will be validated in cell culture for knockdown efficiency (flow cytometry, FACS, qPCR, and protein level) and for potential off target effects (by examining levels of highly homologous genes, if present).

• CRISPR/Cas9 for gene editing and gene expression modulation

  For knockout studies, we will design guide RNAs (gRNAs, using Benchling software or RGEN Tools), insert them into desired vectors (e.g. PX459 v2.0, Addgene) and test their ability to generate in/dels using the SpCas9 nuclease by a T7E1 assay (NEB). Promising gRNA leads will then be prioritized for future in vitro or in vivo experiments. In addition to the conventional Cas9 (e.g. SpCas9), we will provide the option to test smaller variant versions (e.g. CjCas9), which we can use to fit larger transgenes than the SpCas9 allows.

• CRISPR-based epigenetic modulation

  The CRISPR system has been adapted for transcriptional activation, repression, and epigenetic editing by mutations to the catalytic domains of Cas9 to form a ‘dead’ Cas9 (dCas9) protein, which binds the DNA target specified by the gRNA without initiating a double-strand break. Thus, Cas9-directed genomic location targeting can be exploited beyond the DNA double strand breaks. Importantly, dCas9 allows a reversible repression or overexpression of genes, which can circumvent long-term side effects imposed by permanent gene knockout. We will provide the service for designing the gRNAs, as well as validating dCas9 and the transcription repressor (e.g. KRAB), or transcription activator (e.g. VP64) for their research in vitro and in vivo systems.

  The CRISPR-based prime and base editors allow base conversion, as well as small insertions in the DNA. They are attractive tools for therapeutic genome editing because they do not generate double-stranded DNA breaks (DSBs), do not require a DNA donor template, and are more efficient in editing non-dividing cells. Prime editing further expands the scope of base editing and induces a much lower off-target effect than Cas9 endonuclease.

• Differential scale DNA plasmid preparation

  After fully validating the destination constructs by sequencing and restriction digestion (typically mini preparations are sufficient), we will scale up the culture volume to achieve midi preparation scale (for typical transient transfection experiments and adenovirus production), or maxi preparations (for lentivirus production). Qiagen Midi/Maxi Prep Plus kits (which reduce the amount of endotoxin in the resulting DNA) will be used. Confirmation of low endotoxin levels will be confirmed using the Stem Cell, Organoid, and Cell Phenotyping Module. Final DNA concentration will be determined by Nanodrop and verified on a gel, again using restriction digestion.

• Verification of DNA construct expression and correct subcellular localization

  Prior to giving the new DNA construct to the individual investigator, we will verify that it expresses appropriately in basic 293A-based cell cultures. 293A cells will be plated overnight in 6-well dishes to yield enough cellular material for western blotting or qPCR. Cells will be transfected using Lipofectamine 3000. Cells used for western blotting will be lysed in RIPA buffer supplemented with benzonase and protease inhibitor, normalized by BCA and denatured on an SDS-PAGE gel, followed by transfer to a nitrocellulose membrane. Western blotting imaging will be performed on a LI-COR CLx using conventional, validated antibodies to the protein of interest, or an appropriate epitope tag. Cells for qPCR analysis will be processed using an Aurum Total RNA kit. Samples will be reverse transcribed and evaluated for mRNA transcript using TaqMan probes (Life Technologies). Immunolocalization studies will be performed by transfecting 293A cells on glass bottom-plates, followed by fixation, antibody incubation, and imaging on a confocal microscope using the Microscopy and Animal Phenotyping Module. Fluorescent organelle markers (purchased as DNA constructs from Addgene) will be used as positive controls. After validation of these three parameters, constructs, along with all sequencing data and SnapGene files will be provided to the individual investigators for their studies. If mislocalization or poor expression is observed, we will attempt to transfect the construct in a more physiologically relevant cell type (like ARPE-19, 661W, etc.), in case additional protein partners are necessary for production.

• Production/purification of lentivirus and AAVs

  After verification of protein expression, mRNA production and correct subcellular localization, if necessary, replication incompetent lentivirus will be produced using 293T cells with the plasmid of interest, VSVG, and PSP plasmids. Media will be harvested at 48h and 72h and pooled. Lentivirus will then be purified by sucrose gradient ultracentrifugation and viral particles collected. Viral titers are calculated by p24 ELISA (Abcam), as well as functional titers using standard plaque assays/crystal violet staining. Lentivirus will be aliquoted and frozen at -80° in single use aliquots to avoid freeze thaw-mediated reduction in titers.

  Alternatively, AAV will be generated. Upon consultation with the investigator, small- to medium-scale AAV production can be provided (e.g. 50-100 uL in volume with a titer of 1 x 10¹³GC/ml). For larger scale AAV production, the UTSW Translational Gene Therapy Core may be utilized.

• Validation of RNA expression with RNAscope, BaseScope, and MiRNAscope assays

  Spatial analysis of gene expression is an essential validation for plasmids and viruses. Traditional RNA-ISH can be challenging due to low sensitivity and inconsistent detection, accompanied by limited plexing. We will perform or provide training to perform RNAscope assays (ACD), which provide exceptional sensitivity, allowing single-molecule detection of RNA targets at the single cell level. Additionally, we will perform or provide training to carry out BaseScope and miRNAscope assays. The BaseScope Assay enables applications such as the detection of exon junctions/splice variants, short/highly homologous RNA sequences (50-300 bases), and point mutations at single cell sensitivity. It allows highly specific and sensitive detection of RNA targets with down to one nucleotide differences. Detection of small non-coding RNAs such as microRNAs, and short oligonucleotide therapeutics, such as ASO and siRNA require a robust, highly specific, and sensitive assay. While microarrays and PCR both provide useful molecular profiles of diseases, important clinically relevant cell and tissue context information is lost, along with the spatial variation of gene expression patterns. The miRNAscope assay is a highly sensitive and specific in situ hybridization assay that allows for the visualization of ASO, miRNA, siRNA, and other nucleic acid targets between 17-50 nucleotides and expression in intact tissues or cultured cells with single-cell resolution while preserving spatial and morphological context. We will provide the initial consultation for probe design and protocol training as needed and/or execution of the protocol by the Module manager. Each PI will be responsible for providing expendable ACD reagents/supplies not covered by the Module.