In collaboration with Moritz Kueblbeck, Andrea Callegari, Beatriz Serrano-Solano Dr. Jan Ellenberg Group European Molecular Biology Laboratory
Assessing CRISPR-edited mammalian cell lines using a single-step 3-color Crystal Digital PCR™ assay
The generation and usage of homozygous fluorescent knock-in mammalian cell lines using sequence-specific Cas-endonucleases has become a commonly used molecular biology procedure.1-8 Nevertheless, to identify the desired genetic modification and reject clones with off-target events, several hundred monoclonal colonies still need to be screened. The maintenance and screening of high numbers of clones is a laborious, time-consuming, and error-prone process. To facilitate colony screening, we implemented a single-step 3-color assay using Crystal Digital PCR™ to assess on-target copy number and detect off-target events simultaneously. Because digital PCR (dPCR) is highly sensitive, it requires low amounts of biological samples. Thus, the screening assay was integrated early in the genome engineering pipeline. In addition, because the single-step 3-color Crystal Digital PCR™ assay is achieved in a single assay in about three hours, the culture of incorrectly edited clones can be discontinued on the same day and early in the pipeline
1A Donor design (top) | A PCR product, amplified from a synthetic dsDNA template, is used as donor. It includes a short sequence (~50 bp homology arm) homologous to the 5’-region of NUP93 (in grey), a linker (~5-10 amino acids, orange square), a STOP codon and a short sequence (50bp homology arm) homologous to the 3’-UTR of NUP93 (in grey). Notably, the mEGFP sequence contains a silent mutation to generate a restriction to have independent templates during digital PCR (deep blue bar). A STOP codon and a short sequence (50bp homology arm) homologous to the 3’-UTR of NUP93 (in grey). Single-step 3-color Crystal Digital PCR™ assay design (bottom) | Dashed line indicates homology-directed repair (HDR) after the targeted spCas9-mediated double-strand break (DSB). Primer positions are shown to generate products corresponding to “on-target tag,” “total mEGFP,” and “reference” assays. Arrows point to the ratios of products representing total mEGFP copies and on-target mEGFP copies.
1B Crystal Digital PCR™ measurements of six representative clones | Boxes represent on-target (blue) and total mEGFP (green) copy numbers. Every red dot represents an individual dPCR measurement (technical replicates). Allelicity of the NUP93 gene in U-2 OS cells is three. Clones (clone ID 35, 52 and 347) scoring at 3 on-target and at 3 total mEGFP copies are homozygous for NUP93-mEGFP. A total number of mEGFP copies higher than 3 suggests extra integrations (clone ID 25 and 246). Estimates lower than 3 indicate either fewer tag integrations (monoallelic or biallelic clones) or potential genomic rearrangements (clone ID 5).
1C Southern Blot performed with the same clones as in B | The band at ~4.7kb indicates a correctly tagged NUP93-mEGFP allele detected with a mEGFP specific probe. Any additional band detected using the mEGFP-specific probe indicates an extra integration of the tag in the genome. The Nup93-specific probe reveals the correctly tagged NUP93-mEGFP alleles at ~4.7 kb and the untagged alleles at ~4.0 kb. Bands marked with an asterisk correspond to unexpected, extra integrations or major genomic re-arrangements.
2A “On-target-tag” assay detected by the FAM probe (On-target-tag assay) that binds to the junction between the last exon of the endogenous NUP93 editing site and 5’ sequence of the integrated mEGFP transgene.
2B “Total-mEGFP” assay detected by the HEX probe (total-mEGFP assay) that binds to the mEGFP sequence and is designed to detect
only the mEGFP transgene. The reference assay is detected by a Cy®5 probe and binds to a sequence downstream of the target site on
the same chromosome (Chr16).
We have developed a single-step 3-color Crystal Digital PCR™ assay and assessed its predictive power by comparing it to a conventional Southern blot. There are three NUP93 alleles in U-2 OS cells. This quantitative assay showed that clones 35, 52 and 247 scored 3 on-target and total mEGFP copies, meaning that all three NUP93 alleles were successfully tagged (i.e. homozygous clones), with no detectable off-target integrations. The Southern blot confirmed this conclusion. Clone 5 shows an equal number of mEGFP copies for both assays, though without matching the expected number of NUP93 loci. The copy number assessment may indicate a single, correct editing event accompanied by a major genomic re-arrangement, as suggested by Southern Blot analysis. Clone 25 and 246 show a high number of total mEGFP integrations vs on-target integrations, indicating potential off-target editing or incomplete HDR. Southern Blot displays extra bands detected with the mEGFP and NUP93 probes, suggesting genome rearrangements. Results obtained by 3-color Crystal Digital PCR™, a straightforward one-step detection assay, were perfectly concordant with the more labor-intensive southern blot technique.
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