Genome-Editing Design and Analysis Services

CD Genomics offers various solutions, from genome-wide association studies on tens of thousands of samples to single SNP interrogations of several hundred individuals. Our experts and technicians are knowledgeable and have years of experience providing Genome-Editing Design and Analysis Services.

Name of the Instrument/Technicial Service

Genome-Editing Design and Analysis Services

Introduction

Non-Homologous End Joining (NHEJ). We recommend the use of this high-efficiency editing strategy to create large deletions when precise breakpoints are not required.
Homology Directed Repair (HDR) with Single-Stranded DNAs (≤180 nucleotides). We recommend the use of this strategy to introduce small edits, such as point mutations or small protein tags (e.g., HA or Flag), and for the creation of precise DNA deletions.
HDR with Single-Stranded DNAs (181-5,000 nucleotides). This approach enables the modification of longer DNA segments up to approximately 5 kb. It is currently being used to insert loxP sites around one or more exons, to introduce multiple point mutations, and to insert exogenous coding sequences encoding fluorescent proteins or Cre.
HDR with Double-Stranded DNAs (generally > 5 kb). We recommend using double-stranded (ds) when the desired genome edit exceeds 5 kb, or when commercial projection of a long ssDNA is not feasible. We have used this approach to insert two cre-inducible transgenes into Rosa26, and are currently working to improve the efficiency of this approach by using 2-cell homologous recombination.

Protocol

A genome editing strategy is devised and reviewed with the investigator. We have four classes of CRISPR-Cas9 genome editing projects based on the donor DNA format, length of the desired gene edit, and whether the edit is an insertion or replacement.
Guide RNAs are sourced in a crRNA + tracrRNA format and tested for activity in a test tube assay. Guides that pass the test tube activity assay are usually determined to be sufficiently active in vivo, but more complex projects that require mutliple guides may benefit from pre-screening in vivo.
CRISPR editing reagents and a donor DNA, if required, are delivered into the pronuclei of one-cell fertilized mouse zygotes. Our standard strain options is C57BL/6J. Other strains may also be used for genome editing. Inquire for more information.
Resulting pups are biopsied and screened by PCR and Sanger sequencing.
Predicted founders are transferred to the investigator for breeding and validation of the desired genome edit in the N1 generation. Alternatively, breeding and screening of the N1 generation can be performed by VGER.
Investigators are provided with a validated genotyping protocol and a genome editing report that includes a description of what the model is, how it was designed and produced, and additional critical information such as correct nomenclature for the new allele.

CD Genomics has already updated its technology platforms to mainstream NGS, long-read sequencing, and microarray instruments. We continue to strive to offer the same reliable services to pharmaceutical and biotechnology companies, as well as academia and government agencies, for all your sequencing or array needs. If you are interested, please contact us directly for assistance.

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