Journal: bioRxiv

Article Title: Template-independent genome editing and repairing correct frameshift disease in vivo

doi: 10.1101/2020.11.13.381160

Figure Lengend Snippet: a, PCDH15 is one of the two components of the tip link that gates the mechanotransduction channel in hair cells. The 216-kD PCDH15 contains 11 extracellular cadherin (EC) repeats and a transmembrane (TM) domain. The av3j mutation brings an A insertion between the EC11 and TM domain and causes an early stop codon in the Pcdh15 gene. b, Four 17-nt gRNAs were designed to target the av3j A insertion site. The m- 3j -gRNA1, m- 3j -gRNA21, and m- 3j -gRNA24 cover the insertion, while wm- 3j -gRNA9 can target both wild-type (WT) and av3j alleles. c, The editing profiles of the 4 gRNAs shown as the indel frequency at each indel size. The bars of 1-bp deletion were highlighted in red, which was 40.9% for m- 3j -gRNA1. Each gRNA profile had 2-3 replicates. Error bars, SD. d, PCDH15 protein immunostaining in the electroporated P3 av3j/av3j cochleae cultured for 2 days in vitro (P3+2DIV). PX330 containing m- 3j -gRNA1 and Cas9 (PX330-m- 3j -gRNA1) was co-electroporated with N1-EGFP. Note that only the hair bundles of electroporated av3j/av3j hair cells showed obvious signals (arrowhead). Scale bar, 3 μm. e, Averaged traces of Ca 2+ responses activated by fluid-jet stimuli in av3j/av3j OHCs electroporated by control PX330 vector or PX330-m- 3j- gRNA1 (P1+4 DIV). Error bars, SEM. f, Quantification of the maximal Ca 2+ responses in OHCs from recordings similar to ( e ). W- 3j -gRNA1 targets the corresponding WT sequence and shares the same PAM with m- 3j -gRNA1. Responsive and total recorded cell numbers are shown in panels. Brown-Forsythe and Welch ANOVA test; ns, no significance, ****P < 0.0001, error bars, SEM. g, 74.2% (72/97) av3j/av3j auditory hair cells, including both OHCs ( f ) and IHCs (Supplementary Figure 1c), recovered mechanosensitivity after PX330-m- 3j -gRNA1 electroporation. h, 48.7% editing products in m- 3j- gRNA1 are ‘3n-1’, which could restore the reading frame of av3j , and were dominated by 1-bp deletion (D1), 4-bp deletion (D4) and 2-bp insertion (I2) (See Supplementary Fig. 1d for details). Note that, correction of only one allele is sufficient to achieve a functional recovery of the diploid av3j/av3j hair cells.

Article Snippet: The PX330 backbone was digested with BbsI (R0539S, NEB), and ligated with the oligos by T4 ligase (15224017, Invitrogen).

Techniques: Mutagenesis, Immunostaining, Cell Culture, In Vitro, Plasmid Preparation, Sequencing, Electroporation, Functional Assay

Journal: bioRxiv

Article Title: Template-independent genome editing and repairing correct frameshift disease in vivo

doi: 10.1101/2020.11.13.381160

Figure Lengend Snippet: a, Cartoon showing multiple factors relevant for frame-restoration gRNA evaluation: genome editing efficiency, frame-restoration ratios, and editing product function-rescue evaluation. b, Genome editing efficiencies of designed gRNAs, in which the %Indel data come from N2a cell line 72h post transfection of gRNA-carrying PX330 plasmid for it was difficult to tightly control the percentage of electroporated tissue cells throughout FACS sorting. c, The percentage of predicted frame-restored products of designed gRNAs. d, The predicted protein alteration spectrums after genome editing of each gRNA based on deep sequencing data from electroporated tissues. Color code indicates the number of amino acid changes relative to WT.

Article Snippet: The PX330 backbone was digested with BbsI (R0539S, NEB), and ligated with the oligos by T4 ligase (15224017, Invitrogen).

Techniques: Transfection, Plasmid Preparation, Sequencing