Fig 1.
(a) Use of type IIS restriction enzymatic digestion enables rapid cloning of a custom CRISPR gRNA in a plasmid containing Cas9, mCherry and Neo resistance cassettes. Transformants of recombinant plasmids will be white, while transformants of non-recombinant plasmids will be blue. (b) Target cell lines are transfected with the Cas9+gRNA expression plasmid, enriched by cell sorting or antibiotic selection and expanded as single cell clones. (c) Schematic of droplet digital PCR-based screening of successful NHEJ.
Fig 2.
Droplet digital PCR can be used to screen for indels in bulk-edited populations and single cell clones.
(a) Example ddPCR plots of parental population gDNA and bulk-edited population gDNA for three given genomic targets. (b) Conceptual and actual results of a T7E1 digestion resolved by gel electrophoresis for wildtype, mono-allelic mutant and bi-allelic mutant clones. (c) ddPCR plots of wildtype, mono-allelic mutant and bi-allelic mutant clones. Note that FAM and HEX are used for different probes between the SFRP1 and NODAL assays.
Fig 3.
Sensitivity of indel detection in mismatch and ddPCR assays.
(a,b) Genomic DNA samples from wild type and target-disrupted cells were mixed in indicated ratios and subjected to a T7E1 cleavage assay. Gel analysis was conducted on SFRP1 (a) and NODAL (b) targets. DNA sequences of the mutant cellular clone are displayed at the top of the panel. Underlines indicate TALEN spacer region in a, and gRNA target (including PAM sequence) in b. (c-e) Genomic DNA from target-disrupted samples were added to wild type samples at indicated amounts and subjected to ddPCR analysis. Error bars indicate 95% confidence interval. Yellow line depicts the mutant copies expected. “N.D.” indicates none detected.
Fig 4.
Genomic copy number alterations can be detected in ddPCR NHEJ detection assay.
(a) Conceptual diagram displaying the expected changes in reference signal strength dependent on large deletions encompassing the reference probe binding site. (b) SFRP1 reference signal strength (copies/uL) in three C8161 SFRP1-edited cellular clones. (c) Copy number analysis of SFRP1 in three SFRP1-edited cellular clones normalized against RPP30. Error bars represent standard deviation (n = 3).
Fig 5.
Application of ddPCR NHEJ detection assay to determine genome editing efficiencies of NH versus NN RVD-containing TALENs.
(a) TALENs were designed against an SFRP1 target; NH and NN RVDs were used as illustrated. (b,c) Mutant alleles were detected by the ddPCR NHEJ detection assay in bulk population gDNA of C8161 SFRP1-edited cells. Technical (b) and biological (c) replicates illustrate the enhanced gene editing ability of the NN RVD-containing TALEN pair to this target. Points represent percent mutant alleles detected; error bars represent 95% confidence intervals.