Genome editing in the Ellison Stem Cell core
The Ellison Stem Cell Core offers full-service genome editing in pluripotent stem cells from various species (human, non-human primate, mouse). The core provides consultation, design of CRISPR/Cas9 strategies and reagent preparation, as well as cell culture services to test the guide efficiency and screen for edited cell lines. Genome editing services include generation of knock-out (KO), point mutation and knock-in (KI) engineered cell lines.
Generation of KO line using CRISPR-Cas9 system
In order to generate KO, we design sgRNAs targeting the early coding region of the gene of interest using a combination of web-based tools. Particular attention is paid to sgRNA placement within the gene, off-target predictions, and SNP locations. Three to four sgRNA are selected per gene and individually introduced along with Cas9 into pluripotent stem cells using the Amaxa electroporation system through either plasmid or ribonucleoprotein (RNP) delivery. The cutting efficiency of the sgRNAs in the gene of interest is assessed by Surveyor nuclease assay and/or PCR amplification followed by Sanger sequencing.
Once the best sgRNA has been identified, individual clones are hand-picked for clonal expansion and genotyped. Sequencing results are analyzed and screened for indel mutations resulting in frame shifts leading to premature stop codons. Potential heterozygous and homozygous KO lines are identified and topo-cloning of PCR amplicon for those lines is performed to confirm the genotype. At least 1 or 2 homozygous lines are then expanded and cryopreserved to transfer to the investigator. The investigator is responsible for assessing the complete loss of the protein by Western blot analysis, flow cytometry or immuno-fluorescence. Karyotype analysis can be done through Diagnostic Cytogenetics (https://diagnosticcytogenetics.com/cell-line-services/).
Introduction of point mutation using the CRISPR-Cas9 system
In order to recapitulate the specific mutations observed in patients or correct mutations found in patient derived-iPSC, we offer the generation of targeted point (or small) mutations using the CRISPR-Cas9 technology. Following cut by Cas9 enzyme, DNA can be repaired via homologous directed repair (HDR) with introduction of a donor DNA template.
An optimal design of sgRNA is critical for a successful precise gene-editing. We design sgRNA targeting DNA as close as possible to the desired location of mutation (ideally less than 10bp from the mutation). To determine the best predicted sgRNA in our region of interest, we use a combination of web-based tools. Up to 3 sgRNA are selected per gene and individually introduced along with Cas9 into pluripotent stem cells using the Amaxa electroporation system through either plasmid or ribonucleoprotein (RNP) delivery. The cutting efficiency of the sgRNAs in the gene of interest is assessed by Surveyor nuclease assay and/or PCR amplification followed by Sanger sequencing.
Once the best guide has been identified, we design and order a single-stranded oligo donor (ssODN) consisting of about 60bp homology arms on each side flanking the desired mutation. In addition, a silent mutation is introduced in the PAM region of the selected guide on the ssODN, to avoid further cutting of the correct allele by Cas9. Individual clones are hand-picked for clonal expansion and genotyped. The sequencing results are analyzed and clones screened for heterozygous or homozygous lines that incorporated the ssODN template and harbor the desired mutation without any additional Indel. At least 2 genome edited clonal lines are expanded and cryopreserved to transfer to the investigator.
Generation of KI line using CRISPR-Cas9 system
CRISPR-Cas9-mediated KI of exogenous DNA to a selected genomic locus allows efficient generation of luminescent and fluorescent reporter lines. The reporter can be inserted in the endogenous site of the gene of interest or in a “safe harbor” site, such as AAVS1 or Rosa26 locus.
The first steps are similar to the introduction of small mutations (2). We design sgRNA targeting the DNA as close as possible to the desired location of the reporter insertion and up to 3 gRNA are tested for their cutting efficiency. A donor plasmid containing the reporter cassette flanked by homology arms is then constructed. We often introduce an antibiotic-selection marker to facilitate the selection of targeted clones, that can eventually be floxed out later. However, the KI strategies can vary widely depending on the investigator project.
After electroporation of the pluripotent stem cells with the gRNA, Cas9 and donor plasmid, individual clones are hand-picked for clonal expansion. Clones are genotyped and screened for heterozygosity or homozygosity of insertion as well as for undesired additional indel mutations. At least 2 genome edited clonal lines are expanded and cryopreserved to transfer to the investigator.
Notes:
- If the investigator wishes to provide the pluripotent cell line, this line must be tested for mycoplasma prior introduction into the core facility (we recommend using the Mycoplasma Kit- Lonza #LT07-118).
- The detection of off-target mutations is not included in the price. We can however look computationally using existing software for predicted off target regions and design primer pairs to analyze potential mutations at those sites. Additional fees are applied if the investigator wants the core to test those primer pairs and look for mutation in predicted off target sites (by Surveyor assay or Sanger sequencing).
Generation of Cas9 constructs, donor plasmids for KI as well as some of the genotyping is performed through a collaboration with the Diabetes Research Center Vector and Transgenic Mouse Core (https://depts.washington.edu/diabetes/viral-vector-and-transgenic-mouse/).
The Ellison stem cell core also partners with the UW Transgenics facility in order to generate CRISPR-modified mice (http://depts.washington.edu/compmed/transgenic/es_cell.html).
For questions about genome editing services please contact Julie Mathieu at jmathieu@uw.edu.