The Genome Editing Core (GEC) provides 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 gene-edited mouse models to aid in studying the genetic mechanisms underlying diabetes in vivo. In addition, the Core provides services for genome-wide, unbiased, forward screening using lentiviral CRISPR libraries to target mouse or human cells to discover novel genes, pathways, and mechanisms that underlie diabetes and its complications.
These services involve transduction of human or mouse cells of interest with Cas nuclease and guide RNAs (gRNAs) designed to target specific genes across the genome, followed by selection for cell death/survival, cell phenotype, or reporter function. Genetic material is recovered for sequencing to discover relevant guide RNAs and hence, genes of interest.
Table 1. GEC CRISPR libraries
- Screening project consultation. The Core Co-Director, Dr. Peng Yi, and research assistant meet with investigators to gather information on the project’s overall aims and to establish the feasibility of using CRISPR screening to address the particular research question.
- Library selection and preparation. In consultation with The Core maintains stocks for several human and mouse genome-wide lentiviral CRISPR libraries, enabling both gene inactivation (loss-of-function, LOF) and for gene activation (gain-of-function, GOF) screens (table 1). Once the user decides on the most appropriate library for the screen, typically in consultation with the Co-Director, the Core prepares lentiviral particles from the relevant library.
- Cell transduction and selection. Investigators provide the Core with the cell line chosen from initial consultations. The Core research assistant titers the lentiviral library on the cell line provided and then transduces the cells by lentiviral infection (MOI of 0.3-0.6) using freshly prepared lentiviral supernatant. Transduced cells are provided to the investigators to carry out the desired screen and return the screened cells to the core for sequencing.
- Recovery of genomic DNA, sequencing and analysis. The Core extracts genome DNA from screen samples and generates Illumina sequencing libraries. Libraries are sent out for Next Generation Sequencing by a commercial vendor to obtain sequence counts for each gRNA represented in the initial library. Once sequencing results are available, the Core completes initial data processing to annotate and determine the gRNA read counts in the samples.
- Generation of validation cell line. Once a target gene of interest is determined through the screen, the Core can help validate the results by targeting individual loci for follow-up studies. Relevant gRNAs are cloned into a lentiviral vector for CRISPR genome editing, virus is produced and the original cell line is then transduced with the lentiviral preparation.
The Core uses CRISPR/Cas to assist investigators in manipulating 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.
- Mouse genome editing consultation. The Core Co-Director consults with the requesting investigator to optimize design to achieve project goals, develop genotyping strategy, estimate costs, and provide a tentative timeline. The Core leverages its extensive experience with simple gene knockout models, conditional gene alleles, and more complex gene modifications to introduce new genetic elements (e.g., knock-in).
- Construct design. The Core designs gRNA sequences to target the desired genomic region and for projects that entail homology directed repair, corresponding DNA templates are to include the desired gene modifications (e.g., knockin of loxP sequences, fluorescent reporter addition). gRNAs and templates for homology directed repair are ordered and tested by the Core in model cells lines prior to microinjection to ensure the most efficient sequences are used for each project.
- Embryo microinjection. Two separate microinjection sessions are initially performed to deliver several litters of pups to the investigator for genotyping. Additional microinjections are planned depending on the success of the first rounds of injections, estimated first on the number injected and reimplanted embryos, then on the number of live pups born from foster pregnancies, and finally on the number of mutant mice obtained that that are suitable for breeding. The Core offers microinjection into zygotes of any mouse strain (C57BL/6 and NOD mice are most commonly used). The Core handles all mouse husbandry and procedures, obtains ear punch biopsies for genotyping, and returns weaned mice to the investigator.
Remember to Cite the DRC
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."
For questions about forwarding CRISPR screens in cells, email peng.yi [at] joslin.harvard.edu (Peng Yi). For questions about CRISPR modified mice, email Stephan.Kissler [at] joslin.harvard.edu (Stephan Kissler).