Journal: Science Advances
Article Title: Self-inactivating AAV-CRISPR at different ages enables sustained amelioration of Huntington’s disease deficits in BAC226Q mice
doi: 10.1126/sciadv.aea8052
Figure Lengend Snippet: ( A ) Schematic representation of targeted human HTT exon 1. sgRNAs HTTg1 and HTTg2 were designed to target the N17 domain–coding DNA sequence, which is located upstream of the CAG repeats. PAM, protospacer-adjacent motif. ( B ) Schematic representation of AAV-SaCas9-HTT sgRNA vector. U6-driven sgRNA and a CMV-driven Cas9 from S. aureus tagged with HA were packaged into a single AAV vector of 4.5 kb. ITR, inverted terminal repeat; NLS, nuclear localization signal sequence; 3×HA, three tandem repeats of the human influenza hemagglutinin (HA) tag. ( C to K ) Validation of human HTT editing in human embryonic kidney (HEK) 293T cells expressing EGFP-hHTT-20Q or EGFP-hHTT-120Q reporter system. (C) SaCas9-HTTg1 and SaCas9-HTTg2 reduced EGFP-fusion protein expression and aggregation in cells, whereas the control SaCas9-sgRNA did not. Scale bar, 100 μm. [(D) and (E)] Quantitative analyses of EGFP-20Q (D) or EGFP-120Q (E) fluorescence intensity. n = 3 or 4; ** P = 0.0083 (20Q), ** P = 0.0087 (120Q), and *** P = 0.0009 (120Q). [(F) to (K)] Western blot detecting EGFP and SaCas9 (anti-HA) protein level in EGFP-hHTT-20Q [(F) to (H)] or EGFP-hHTT-120Q [(I) to (K)] reporter system. [(F) and (I)] SaCas9-HTTg1 and SaCas9-HTTg2 reduced EGFP-hHTT protein expression compared to control SaCas9-sgRNA with equivalent SaCas9 expression level. [(G), (H), (J), and (K)] Quantification of relative EGFP [(G) and (J)] and SaCas9 [(H) and (K)] protein expression. n = 3 per group; ** P = 0.0087 (20Q) and ** P = 0.0069 (120Q). Data were shown as means ± SEM. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.
Article Snippet: A single-vector AAV-SaCas9 system containing Cas9 from Staphylococcus aureus (SaCas9) and its sgRNA scaffold was obtained from Addgene (plasmid no. 61591). sgRNAs targeting human HTT and SaCas9 were designed on the basis of PAM sequence (5′-NNGRRT-3′) and by CRISPR RGEN Tools ( www.rgenome.net/ ). sgRNA sequences targeting human HTT are as follows: HTTg1: 5′-TGG AAA AGC TGA TGA AGG CCT-3′; and HTTg2: 5′-GAA AAG CTG ATG AAG GCC TTC-3′.
Techniques: Sequencing, Plasmid Preparation, Biomarker Discovery, Expressing, Control, Fluorescence, Western Blot
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![( A ) Schematic representation of targeted human HTT exon 1. sgRNAs HTTg1 and HTTg2 were designed to target the N17 domain–coding DNA sequence, which is located upstream of the CAG repeats. PAM, protospacer-adjacent motif. ( B ) Schematic representation <t>of</t> <t>AAV-SaCas9-HTT</t> <t>sgRNA</t> vector. U6-driven sgRNA and a CMV-driven Cas9 from S. aureus tagged with HA were packaged into a single AAV vector of 4.5 kb. ITR, inverted terminal repeat; NLS, nuclear localization signal sequence; 3×HA, three tandem repeats of the human influenza hemagglutinin (HA) tag. ( C to K ) Validation of human HTT editing in human embryonic kidney (HEK) 293T cells expressing EGFP-hHTT-20Q or EGFP-hHTT-120Q reporter system. (C) SaCas9-HTTg1 and SaCas9-HTTg2 reduced EGFP-fusion protein expression and aggregation in cells, whereas the control SaCas9-sgRNA did not. Scale bar, 100 μm. [(D) and (E)] Quantitative analyses of EGFP-20Q (D) or EGFP-120Q (E) fluorescence intensity. n = 3 or 4; ** P = 0.0083 (20Q), ** P = 0.0087 (120Q), and *** P = 0.0009 (120Q). [(F) to (K)] Western blot detecting EGFP and SaCas9 (anti-HA) protein level in EGFP-hHTT-20Q [(F) to (H)] or EGFP-hHTT-120Q [(I) to (K)] reporter system. [(F) and (I)] SaCas9-HTTg1 and SaCas9-HTTg2 reduced EGFP-hHTT protein expression compared to control SaCas9-sgRNA with equivalent SaCas9 expression level. [(G), (H), (J), and (K)] Quantification of relative EGFP [(G) and (J)] and SaCas9 [(H) and (K)] protein expression. n = 3 per group; ** P = 0.0087 (20Q) and ** P = 0.0069 (120Q). Data were shown as means ± SEM. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8519/pmc12998519/pmc12998519__sciadv.aea8052-f1.jpg)