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Table 1 Sequencing allows further refinement of zygosity

From: A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality

  1. Sequence analysis of specific clones to verify the status of EZH2 alleles. In both of the examples from the G-401 cell clones (A and B), one allele of EZH2 is disrupted by a deletion (16 bp in clone A and 47 bp in clone B) and the other allele in both clones has a 1 bp mutation that disrupts the Fau1 site. Both alleles have been edited and the restriction digest in these clones correctly indicated a homozygous disruption: the fragment length PCR analysis also indicated correctly the presence of an allele of undisrupted length. The sequencing data shows that in all probability these clones have retained a functional EZH2 allele owing to an in frame mutation. In both of the KYM-1 clones (C and D), both alleles have been edited. Again, the digest data correctly indicated homozygous edited alleles for both clones. For clone C, the fragment length analysis indicated heterozygous edited alleles which was confirmed by the sequencing data showing one allele with an in frame 3pb alteration. Clone D from KYM-1 cells showed both fragment lengths corresponded with an un-edited fragment length, and this was confirmed by the sequence data, which showed a 1 bp alteration in both alleles. This explains why the restriction digest indicated homozygous edited alleles because the restriction digest site is mutated in both cases. These data also suggest that EZH2 remains expressed in these clones