The Genome Editing Core (GEC) provides Joslin Diabetes Research Center (DCR) investigators with resources to manipulate the genome of laboratory mice and cells with the aim of studying genetics underlying diabetes and its complications.
The GEC maintains a facility for the generation of transgenic mouse models to aid in studying the genetic mechanisms underlying diabetes in vivo. The mouse modeling facility uses CRISPR/Cas9 or lentiviral RNAi constructs to introduce genome modifications to a variety of mouse lines in order to establish new models of the disease within a living mammalian system.
In addition, the GEC recently started offering CRISPR screening services. The core acquired several genome-wide lentiviral CRISPR libraries that are now available to target mouse or human cells and to perform unbiased forward genetic screening.
This Core enables investigators to manipulate the genome of laboratory mice in order to generate the most relevant experimental models to understand the genetics and mechanisms underlying diabetes and its complications. The GEC blends Joslin's research expertise in mouse models for diabetes and the generation of genetically modified mice.
The GEC will support the production of genetically modified mice, tailored to investigators' experimental questions by performing pronuclear and intracytoplasmic microinjections. If necessary, the Core can also provide lentiviral transgenesis services for generating mice that carry transgenes mediating inducible or constitutive silencing of target genes of interest by RNAi. The primary method of genetic modification is the CRISPR/Cas9 complex.
The Core will perform mouse genome editing using a variety of targeted edits made possible through CRISPR/Cas9. This includes creating knockout mice by generating a random mutation following a double stranded break (DSB) and non-homologous endpoint joining (NHEJ). For more specific modifications, the use of DNA templates can effect sequence insertion or replacement for knock-in models, for example. Other methods of influencing gene activation or repression are available and can be used upon request.
- Generation of CRISPR-modified mice. GEC director and staff will aid in the design and preparation of guide RNA and DNA templates for sequence deletion or replacement. Investigator-provided materials will be combined with GEC-provided Cas9 mRNA or protein and injected in GEC-provided C57BL/6 and NOD zygotes. Investigators may provide a different strain of mouse, if necessary.
- Consultation on design of gene-modified mice. The GEC will provide advice on construct design for both CRISPR-Cas9 genome editing and lentiviral RNAi. Consultations with investigators will ensure that all researchers use standardized, GEC-approved protocols for preparing injection reagents.
- Consultation on CRISPR gRNA design and testing. The GEC will share its experience with CRISPR/Cas9 to aid in the design of efficient, on-target sequences and of strategies for sequence replacement or gene knock-in. Advice on the use of certain in vitroassays will be provided when requested.
The CRISPR Screen Core aims to help researchers to take advantage of the cutting-edge whole-genome CRISPR screen technology to do innovative research and discover new targets for your research projects.
The CRISPR Screen Core can help design, execute, and analyze genome-wide CRISPR/Cas9 based knockout or overexpression screen in human or mouse cells. The Core can also help generate mutant cell lines through CRISPR technology for validation and follow-up studies.
The specific services of the CRISPR Screen Core include:
- Consultation: The Core director and staff will assist the researcher in determining the feasibility of the project, selecting appropriate cell types and CRISPR libraries, and developing an overall optimal screen
Genome-wide CRISPR libraries currently available in the Core (All obtained from Addgene):
Human GeCKO V2 knockout library (1 plasmid system, 123411 gRNAs)
Human Brunello knockout library (1 plasmid system, 76441 gRNAs)
Human SAM V2 overexpression library (2 plasmid system, 70290 gRNAs)
Mouse GeCKO V2 knockout library (1 plasmid system, 130209 gRNAs)
Mouse Brie knockout library (1 plasmid system, 78637 gRNAs)
Mouse SAM V1 overexpression library (3 plasmid system, 69716 gRNAs)
- Lentiviral library production and cell transduction: The core will prepare lentiviral library propagated using the plasmid CRISPR libraries listed above, titer the lentiviral library and transduce the cells of interest.
- Genome DNA preparation and sequencing: The Core will extract genome DNA from screen samples, amplify and barcode the gRNA region and prepare Illumina sequencing library. Next generation sequencing will be done through a specialized sequencing company, and the Core will provide basic data analysis.
- Generation of validation cell line: Once a target gene of interest is determined through the screen, the core can provide service of individual gRNA cloning, lentiviral production, and mutant cell line generation for validation.
If any of your research has been supported in full or in part by our core, please acknowledge our NIH/NIDDK grant as follows: "Supported by the Genome Editing Core of NIH P30 DK036836."
If you have questions about our core, iPSCore [at] joslin.harvard.edu (email us) directly. For questions about CRISPR modified mice, email Taylor.Roberts [at] joslin.harvard.edu (Taylor Roberts) or Stephan.Kissler [at] joslin.harvard.edu (Stephan Kissler).