Fig. 2.
oaHDR- or HITI-mediated gene knock-in profile after SATI mediated gene-correction of progeria mice in vitro and in vivo. a Schematic representation of the LmnaG609G (c.1827C>T) gene correction with SATI-mediated gene-correction donor. Red box indicates exon 11 with single point mutant. After gene correction mediated by NHEJ-mediated HITI, targeted sequence including corrected mutation are inserted in intron 10, just upstream of mutated exon 11 (left). After gene correction mediated by oaHDR, the mutation is corrected with no change of other genomic sequence except for point mutation (right). The expression level of Lamin C transcribed from exon 1–10 is not affected by Lmna c.1827C>T mutation. After gene correction, Lamin A protein is expressed instead of Progerin expression. Pink pentagon, Lmna intron 10 gRNA target sequence. Yellow scissors or Black line within gRNA target sequence, Cas9 cleavage site (see also Supplementary information, Fig. S8a). b The ratio of HITI, oaHDR, and undetermined (due to large deletion) in targeted sequence after SATI mediated gene correction from progeria MEF (top panel, n = 48), primary neuron (middle panel, n = 47), and brain (lower panel, n = 19). Actual knock-in ratio is indicated in the graph (%). c The ratio of HITI, oaHDR, and undetermined (due to large deletion) with or without indel at targeting site after gene correction by Cas9/Lmna-gRNA-mCherry/MC-Progeria-SATI transfection with shRNA gene knock down for progeria MEFs. Actual targeting ratio is indicated in the graph (%). Each target of shRNA knockdown is indicated at bottom. Scramble control, n = 48; Ku80, n = 19; Lig3, n = 32; Rad51, n = 17. d Experimental scheme for in vivo gene correction by AAV-Progeria-SATI via intravenous (IV) AAV injections to LmnaG609G/G609G progeria mouse model. AAV-Progeria-SATI is injected into newborn (postnatal day 1, P1) mouse together with AAV-Cas9. The phenotypes are analyzed in the indicated date in each experiment. e Gene correction efficiency at Lmna c.1827C>T dominant point mutation site from the indicated tissues in SATI-treated (Pro+SATI) or only donor-treated without Cas9 (Pro+donor) progeria mice at day 100. f Indel percentages at Lmna intron 10 gRNA target site from the indicated tissues in SATI-treated (Pro+SATI) or only donor-treated without Cas9 (Pro+donor) progeria mice at day 100. g The ratio of HITI, oaHDR and undetermined (due to large deletion) with or without indel at targeting site after gene correction by systemic AAV-Progeria-SATI injection for progeria mice. Deep sequencing was performed using the extracted DNA from liver (top) and heart (bottom), respectively. Actual knock-in ratio is indicated in the graph (%)