CRISPR-Cas9 Line Development


CRISPR-Cas9 Founder Identification and Line Development Planning

CRISPR-Cas9 is a rapid, simple, and efficient gene editing method used to create mutations in established lines, and in multiple genetic backgrounds, in a fraction of the time of previous methods. However, founder animals are often mosaic with more than 1 mutation at loci of interest. This mosaicism leads to the most vivarium level confusion and trouble interpreting sequencing results. Before these new lines are viable for your research, careful genotyping and breeding of the founders is essential to ensure the genetics are correctly identified, documented, and understood. This requires meticulous attention to detail in both the laboratory and the vivarium. Our founder characterization and line development services include three essential steps to facilitate this entire process.

CRISPR-Cas9 Founder Identification and Line Development Planning Process

Step 1.

We design a loss of allele assay and screen your initial founders to differentiate animals with 0 vs 1 vs 2 copies of the targeted gene.

You provide:

  • CRISPR mutation experiment information including information on the guide RNA sequences and the expected site of the mutation
  • Tissue samples from all potential founder animals you wish to have assessed (typically 20-30)

Deliverables:

  • Loss of allele assay design
  • Loss of allele results for submitted samples

Pricing:

  • Loss of allele assay design: $595/assay
  • Loss of allele screening: $45/sample

Step 2.

We screen positive founders by direct sequencing to determine mutation elements.

You provide:

  • May need additional tissues samples from identified founders

Deliverables:

  • Genomic sequence for up to five founders
  • Nature of Crispr-Cas9 directed mutations found for each sample
  • Nomenclature of alleles (assigned by GTCA) to be used as standard vocabulary in further genotyping
  • Founder line breeding plan with color guided delineation of founder lines, expected progeny outcomes, and progeny genotyping recommendations to confirm line genetics

Pricing:

  • Sequencing, data analysis and genetic impact of mutation: $175/sample

Step 3.

We screen progeny from founder line matings by direct sequencing to determine mutation elements.

You provide:

  • Tissue samples from N1 progeny animals with assigned mutations from founder line

Deliverables:

  • Sequencing results and recommendations of which mice to proceed with
  • Design and validation of probe-based allele-specific genotyping assay(s) for the mouse line(s)
  • A comprehensive report, including all test results, for documentation of founder line establishment

Pricing:

  • Sequencing: $175/sample
  • Allele-specific assay design: $500/assay
  • Genotyping: $10/sample


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