Fig. 4

Induction of a frameshift mutation in ALMS1 with CRISPR/Cas9 gene editing. A Workflow from gene edition to single cell-derived clone semi-automated isolation and amplification. B Schematic representation of the targeted area (exon 3 of ALMS1 gene) by CRISPR/ Cas9 with the selected gRNA. PAM = protospacer adjacent motif. C Quantification of the percentage of indels following CRISPR/Cas9 according to the mode of transfection. Electroporation method leads to a higher rate of indel events within the cell population compared to lipofection. D Evaluation of the percentage of indels according to the nucleotide position. (0 corresponds to the Cas9 cleavage site). E Evaluation of the probability of nucleotide insertion according to the type of transfection based on sequencing results. F Comparison of the predicted amino acid sequence of ALMS1 protein in WT cells and KO purified clones (with + 1 bp insertion). Following gene edition, the sequence of amino acids is modified (red letters) and a STOP codon appears quickly downstream of the insertion site (red star). G Sequences of the site of gene edition before CRISPR/Cas9 editing (wild type); following edition in a representative edited cell population (knock out pooled cells), and in a representative single cell-derived clone using the semi-automated method. The gRNA is underlined in black and the PAM motif in red. The dotted black line corresponds to the Cas9 cleavage site. Mean of 3 independent experiments