Genome Engineering

MillerLab_Washington_University_Pipe1

The CRISPR/Cas9 system includes the Cas9 nuclease, which can be programmed to target and cleave specific sequences in the genome through the use of a guide RNA (gRNA) that directs it to any user specified position in the genome. The Cas9 protein binds to the site specified by the gRNA and produces a targeted double-strand break in the DNA.  This break is then repaired by the cell and can result in deletions and/or insertions resulting in gene inactivation. Alternatively, if a homologous template is also introduced, repair can occur via homologous directed repair resulting in precise, user-defined gene editing. This allowsinvestigators to produce mutant cells by modifying a single or many nucleotides in a defined fashion.

Custom, validated CRISPR/Cas9 vectors  

Multiple backbones are available (e.g. separate Cas9 and gRNA backbone, all-in-one Cas9/gRNA backbone, all-in-one Cas9-GFP backbone, etc)

  • CRISPR nuclease design
    • We do a full bioinformatics workup for each target that includes an off-target analysis, SNP check, and recommendations for gRNA placement within your region of interest.
  • CRISPR nuclease assembly and validation
    • We prepare two gRNA plasmids and functionally test them for each project. We introduce the CRISPR nuclease into cells and use our validation assay to demonstrate that the CRISPR nuclease is cutting the desired endogenous, chromosomal target site. We only charge for a project after we have successfully identified at least one active CRISPR nuclease.
  • Deliverables – 3-4 week turnaround time
    • Plasmids containing the gRNAs and the Cas9 expression plasmid
    • The primers used in validation assay to detect CRISPR nuclease activity.

CRISPR nuclease mRNA production that is ready for embryo injection

CRISPR nucleases have successfully been used to create novel mouse and rat transgenic models. Direct injection of mRNA encoding Cas9 and the gRNA into one-cell stage embryos results in rapid and efficient generation of model animals with a wide variety of modifications including gene deletions, point mutations, conditional knockouts, epitope and fluorescent reporter gene tagging and multiplex modifications. Although, AMRF investigators are primarily utilizing rodent models, most other organisms can be modified using CRISPR/Cas9 methods because the technology is species-agnostic.

  • CRISPR nuclease mRNA production
    • We synthesize both the Cas9 mRNA and gRNA in a form that is ready for direct embryo injection.
  • Deliverables – 2 week turnaround time
    • gRNA at >300ng/ul concentration
    • Cas9 mRNA at >100ng/ul (14ug total)

Custom Cell Line Creation – Prices vary depending on project. Please contact us to get a quote.

We use CRISPR nucleases to create custom cell lines. Projects are divided into several phases and billed upon the completion of each phase (see Table 1).

  • Specific mutations can be engineered into most cell lines.
  • Custom modifications include but are not limited to:
    • Gene knockout
    • Gene knockin
    • Single nucleotide or codon swaps
    • Protein tagging
    • Small or large genomic region deletions
    • Conditional (flox or frt) modified alleles

Custom iPSC Line Creation – Prices vary depending on project. Please contact us to get a quote.

CRISPR nucleases can be used to create iPSC lines with specific genomic modifications (as above). The capability to genetically incorporate (or correct) disease-causing point mutations in patient-derived iPSC lines will be invaluable for elucidating disease mechanisms through functional genomics, for identifying therapeutic agents and for the development of new cell-based therapies. The GEC has now been merged with the Induced Pluripotent Stem cell (iPSC) facility, which banks patient fibroblasts, generates iPSCs, and develops iPSC differentiation schemes. This allows for a seamless transition from patient derived iPSC generation to the engineering of modified iPSC lines. The GEP will have access to all the capabilities of this newly combined Center.

Projects to develop custom iPSC lines are divided into several steps with phases (Table 1) and billed upon the completion of each phase:

 Donor design and creation services – Prices vary depending on project. Please contact us to get a quote.

To produce user-defined knockin mutations (e.g. incorporation of disease-causing point mutations), a donor substrate (either DNA fragment or oligonucleotide) must be co-delivered with the CRISPR nuclease and incorporated into the locus by homology directed repair. We offer donor plasmid and donor oligonucleotide design and validation services in addition to the requisite CRISPR nucleases.

  • For each knockin project, a GEiC scientist will discuss your overall project goals and design appropriate plasmid or oligonucleotide donors.

 Donor validation services 

Once a donor substrate and functional CRISPR nuclease have been created, we use one of our well-characterized “core” cell lines (e.g. human K562, mouse N2A) to confirm that the donor and CRISPR nuclease are creating the desired modification. Depending on the functional assay used to validate your modification, we can often give you an approximate frequency of the modification.

Table_1_Cell Line Creation PhasesTable 1: Cell Line Creation Phases: Evaluation phase – Prior to taking on a custom cell line project, we test several key criteria before moving forward. We optimize transfection efficiency (typically by nucleofection), we determine the rate of homology directed repair in the desired cell line, and we determine if the desired cell line can grow out from a single cell clone. Phase 0 – CRISPRNucleases are designed, assembled, and validated using our mismatch detection assay in one of our “core” cell lines. If the project involves a donor substrate (i.e. making a point mutation or adding epitope tag), we design and assemble the donor substrate and validate that the desired modification is occurring in one of our “core” cell lines. Phase I – The reagents that have been validated in Phase 0 are used for transfection in the researcher-defined cell line. The phase is complete once the desired modification has been detected in the transfected cell pool. Phase IIA – Single cell dilution cloning is performed to generate pure single cell derived clones, these cells are expanded, and then screened for the desired modification. We will screen up to 500 clones. Phase IIB – Positive clones are expanded and the genotype is reconfirmed prior to clone cryopreservation. Phase IIC – Prior to shipping the modified cells, we perform a test for mycoplasma infection. In the case of modified pluripotent stem cells, they are also monitored for expression of pluripotent markers and tested for chromosomal abnormalities to ensure, to the best of our ability, that the cells retained pluripotent potential throughout the genome engineering procedure.