Fig. 3.

Reversion of Aicda ^−/− CH12F3 clone by knock-in. a Restoration of the Aicda gene in a clone in which both alleles of the genes were inactivated (5bis), either by a 26 bp or a 1 bp deletion. The allele with the 1 bp deletion was targeted with an sgRNA (PAM sequence indicated by the green box) and a single-stranded 100-mer DNA fragment (ssDNA) bearing both the base to be inserted (red) and a mutated PAM sequence (blue). The mutation on the PAM served as marker to distinguish the restored sequence from potential contamination by wild-type Aicda. Sequencing of clones selected with our enrichment system in which CSR was restored (restored clones) confirmed the knock-in of the restoring mutation in most clones. All clones bear the allele with the 26 bp deletion. b Representative FACS analysis of Class Switch Recombination (CSR) in Aicda ^−/− CH12F3 clones targeted for restoration of the Aicda gene. The population of cells expressing IgA is gated in the red box. CSR levels for wild-type (wt) and Aicda ^−/− cells are shown. Representative plots for two independent clones in which CSR was restored are shown (Aicda ^−/restored). The efficiency of CSR in restored clones is diminished compared to the wild type because only one allele has been targeted. c Knock-in efficiency in Aicda ^−/− CH12F3 cells using either sgRNA/Cas9 alone (Cas9), sgRNA/Cas9 coupled with the ssDNA fragment (Cas9 + ssDNA), or the sgRNA/Cas9 and the ssDNA fragment together with the pBSR plasmid (Cas9 + ssDNA + pBSR). The bar diagram on the left shows the total number of clones that were either proficient or deficient for CSR (overall number of clones: Cas9, 33; Cas9 + ssDNA, 50; pBSR + Cas9 + ssDNA, 21). The bar diagram on the right shows the percentage of clones in which CSR was restored. The error bars indicate the SEM from three independent experiments. There is no statistical difference among the various groups