Figure 1.

Development of gene editing approach for functional characterization of SZT2 VUSs. (A) Individual cell clones either homozygous or heterozygous (+ loss-of-function on other allele) for an individual SZT2 variant were generated by gene editing followed by limiting dilution cloning. Immunoblot for amino acid sensitive mTORC1 activity by P-S6K levels was used to determine whether individual variants caused SZT2 loss-of-function. Homology-directed repair rate of cells generated by transfection of px459 encoding targeting gRNAs was analysed using amplicon sequencing and varied between 12% and 25%. (B) Immunoblot of mTORC1 activity in control HEK cells (i.e. unedited) and homozygous HEK SZT2^{p.Val1984del/p.Val1984del} clone. (C) Densitometric quantification of (B). At least three individual replicates were performed. *P < 0.05 (t-test).