CRISPR/Cas editing

Artists rendering of CRISPR Cas9 technology with RNA guide and DNA depictedNishimasu et al., Cell 2014

Overview

We use the latest available CRISPR/Cas technologies to generate small modifications such as indel-knockouts, deletions, point mutations and conditional knockouts; as wells as insertion of large knockins such as reporters or exogenous expression cassettes.  Depending on the modification required we will recommend the best targeting approach.  We support all steps of CRISPR projects including:

  • Targeting strategy and design
  • gRNA selection and validation of efficiency
  • Oligo template design and synthesis order
  • Construct design and cloning
  • Zygote microinjection or electroporation with DNA/RNA/Cas9 protein
  • Cell line transfection via lipofection or electroporation.
  • Isolation of edited cells by FACS. Expansion and screening of clonal cell lines.
  • PCR genotyping strategy and optimization, genotyping and validation of GE clones or offspring by T7E1 assay and sequencing

Currently, we support in-house CRISRP/Cas editing for rats, mice, and cell lines.  We provide consultation for editing in zebra fish, mosquito and other species.  Please contact us if you are working with a different experimental system and need assistance.

Service details:

For simple modifications (indel knockout, deletion, point mutation) we use one or two gRNAs, and a donor oligo when a repair template is required.  for targeting and the service fee includes manipulation of 100 embryos or generation of 5 founders, whichever comes first. If more embryos are required to achieve a modification, additional costs may apply.

For cell lines we support FACS sorting, single clone expansion and screening of two 96 well plates per gRNA.

For conditional knockouts we currently require two rounds of targeting to insert LoxP sites successfully on the same allele, if feasible we can also use a construct template to generate a replacement (those will be treated as a large insertion projects). We perform gRNA validation for these projects, to increases chances of correct repair.

For large insertions, we currently generate a template construct with homology arms and use it in conjunction with CRISPR.  As other methods such as HITI become available to increase efficiency of CRISPR-mediated knockins, we will incorporate those into our workflow. We perform gRNA validation for these projects, to increases chances of correct repair.

For generating genome edited mouse lines on a background strain other than C57BL/6, FVB/N, CD1 or rat lines on a strain background other than SAS (Sprague Dawley) or Fischer F344, additional animal costs may apply.

We require that newly generated founder and chimera animals be transferred to the requesting investigator laboratory within two weeks of transfer approval and serology testing (if required).  If you require any additional services to be performed on the animals, per diem charges for animal housing and care by TGEF will be incurred.