Journal: Nature biotechnology

Article Title: Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing

doi: 10.1038/nbt.4005

Figure Lengend Snippet: In vivo delivery of chemically modified sgRNAs and Cas9 mRNA induced knockout of targeted gene in the mouse liver. (a) Cas9-2A-GFP transgenic mice were injected with one or two doses of sgRNA encapsulated in lipid nanoparticles (LNP). For mice treated with two doses, the second dose was given 5 d after the first dose. (b) Liver were taken 10 d after first dose. Indels at GFP locus in total DNA from liver were measured by TIDE analysis. (c) C57BL/6 mice were i.v. injected with two e-sgRNAs targeting Pcsk9 and Cas9 mRNA encapsulated in LNP. (d) The serum Pcsk9 levels. (e) The serum cholesterol levels. (f) The gene editing events at Pcsk9 locus in total liver DNA, illustrated by deep sequencing. (g–j) C57BL/6 (g,h,j) and FAHmut/mut (i) mice were i.v. injected with one of native, 5′&3′ and e-sgRNA targeting Pcsk9 (g,h) or Fah (i) or ROSA26 (j) and Cas9 mRNA encapsulated in LNP. Indels at Pcsk9 (g,h), Fah (i) and ROSA26 (j) loci in total DNA from liver were measured by TIDE analysis. (n = 6 mice in the e-sgRNA/one dose group of b, n = 5 in Fig. 4j and n = 4 mice in others) *P < 0.05, #P < 0.05 compared to the one dose e-sgRNA-treated group, error bars as s.e.m.

Article Snippet: The Pcsk9 sgRNA sequences were designed according to sgRNA designer software published by the Broad Institute.

Techniques: In Vivo, Modification, Knock-Out, Transgenic Assay, Injection, Sequencing

Journal: Nature biotechnology

Article Title: Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing

doi: 10.1038/nbt.4005

Figure Lengend Snippet: GUIDE-seq genome-wide off-target analysis of nuclease activity for SpCas9 programmed with PCSK9-1 or PCSK-2 sgRNA expressed from a U6 promoter (plasmid), unmodified, end-modified (5′&3′) or e-sgRNA. (a) The bar chart indicates the number of off-target peaks detected in the GUIDE-seq data for each type of sgRNA. (b) The bar chart indicates the fold improvement in Specificity Ratio (SR) (number of unique reads at the target site/sum of the unique reads at all off-target sites) for each sample relative to the plasmid expressed sgRNA. (c) The Venn diagram displays the distribution of the number of GUIDE-seq identified off-target sites that are common or unique for each given treatment group for PCSK-2. For the ten off-target sites unique to the e-sgRNA, nine of these sites have peak scores less than 16, which indicates that they are either weak sites or false positives. For reference the target site score is 9,484 (Supplementary Table 3). (d) Off-target sites obtained from GUIDE-seq were amplified using liver samples from mice treated with LNP-encapsulated Cas9 mRNA, PCSK9-1 and PCSK9-2 sgRNA. Deep-seq was performed to determine mutation frequency. The bars indicate total indel frequencies at the off-target sites. n = 3 mice, error bars as s.e.m.

Article Snippet: The Pcsk9 sgRNA sequences were designed according to sgRNA designer software published by the Broad Institute.

Techniques: Genome Wide, Activity Assay, Plasmid Preparation, Modification, Amplification, Mutagenesis