In Vitro and In Vivo Assay for CRISPR/Cas9
Activity of CRISPR/Cas9
DNA: + indicates genomic DNA from JM8A3 ES cells transfected with 15 ug PNm pX330 plasmid and 5 ug PGKpuro plasmid placed under 2ug/ml puromycin selection for two days, and cultured another four days before DNA extraction.
5R1 and 5R2: pX330 plasmids targeted to 5’ side of exon.
3R1 and 3R2: pX330 plasmids targeted to 3’ side of exon.
Cel I: + indicates addi*on of Cel I enzyme to PCR product.
MT: empty lane.
Arrowheads indicate DNA fragments produced by Cel I cleavage aXer mutant and wild type DNA strands present in PCR product were denatured and annealed.
Activity of Endonucleases
Fertilized mouse eggs were microinjected with endonucleases to cut genomic DNA. Eggs were cultured to the blastocyst stage. PCR was used amplify a DNA surrounding the cut site. The PCR product was purified from an agarose gel and submitted for DNA sequencing. Blue shading indicates sgRNA sequence.
Panel A: DNA sequencing chromatogram showing only wild type genomic DNA is present in the blastocyst.
Panel B: DNA sequencing chromatogram showing multiple DNA sequences are present in the blastocyst. This indicates that nuclease cut the chromosome and the egg repaired the damage by non-homologous endjoining.