Additional file 1 of Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes

Additional file 1: Figure S1. Generation of polyclonal LIG4 knock-out HAP1 cells. A. Schematic diagram of the LIG4 DNA region targeted by the gRNA, and localization of the AflIII restriction site used to knock out this gene in HAP1 cells. B. Schematic diagram of the protocol used to generate the polyclonal LIG4 KO cell line. C. Electrophoresis results following digestion with the AflIII restriction enzyme for screening of the clones. D. Sanger sequence alignments of the clones selected on basis of AflIII restriction site loss relative to HAP1 parental cells. E. Sanger electrophoregrams of LIG4 KO clones with insertions or deletions causing frameshifts relative to the HAP1 parental cell line. Figure S2. Analysis of coverage and frequencies of insertions and deletions obtained following genome editing and NGS sequencing of BRCA1 and BRCA2 variants. A. Insertion and deletion frequencies determined following the NGS sequencing of BRCA1/2 variants of interest, compared with those for the corresponding silent control. These means include the 7 nt surrounding the mutations of interest. B. Analysis of NGS sequencing coverage with the following formula: Mutation of interest (Mutation+Reference control)/Silent control (Mutation+Reference control). Results for all BRCA1/2 variants characterized by comparison with the classified benign and pathogenic mutations (Mann-Whitney tests, p = 0.1934 and p = 0.2902). C. Frequencies of insertions and deletions according to the NGS sequencing of BRCA1/2 variants and corresponding silent editing controls and linear regression analysis. These means include the 8 nt surrounding the PAM sequence. Variants classified in databases are shown in colours: benign mutations in green and pathogenic mutations in red. Figure S3. Evaluation of the reproducibility of our functional assay. A. Replicates of functions scores from four different variants obtained by following the described protocol in the LIG4 KO HAP1 cells (n = 3). Among these, the variants p.Pro1812Ala and p.Tyr422X were already classified as pathogenic in databases, the other two were VUS or had a conflicting interpretation. B. Sanger electrophoregrams of XRCC4 KO clones with insertions or deletions causing frameshifts relative to the HAP1 parental cell line. Clones 5 and 17 were pooled to generate the polyclonal XRCC4 KO HAP1 cell line. C. Comparison of the functional scores evaluated after CRISPR-Cas9 editing and NGS sequencing of four BRCA1 variants in polyclonal LIG4 KO cells and polyclonal XRCC4 KO cells. Table S1. gRNA and oligonucleotides sequences designed to edit BRCA1, BRCA2 and POLE variants in the study. Table S2. Odds of Pathogenicity (OddsPath) estimated by performance of classified variant controls. In this assay, five benign and five pathogenic variants of BRCA1 and BRCA2 were used as controls allowing the estimation of the evidence strength of our classification according to Brnich et al [34] guidelines. Table S3. Variants used as silent controls or references with their classification according to different databases. Benign mutations are shown in green (+), unreported mutations are shown in yellow (?), mutations with conflicting interpretation are shown in orange (+/-), pathogenic mutations are shown in red (-) and intermediate mutations are shown in blue (I).