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Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs.

Gu S, Zhang Y, Jin L, Huang Y, Zhang F, Bassik MC, Kampmann M, Kay MA - Nucleic Acids Res. (2014)

Bottom Line: The reduced off-targeting was confirmed by RNA-Seq analyses from mouse liver RNAs expressing various anti-HCV shRNAs.Compared with previously established work, the new algorithm was more effective in reducing off-targeting without jeopardizing on-target potency.These studies provide new rules that should significantly improve on siRNA/shRNA design.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.

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Related in: MedlinePlus

Silencing efficacy and off-targeting of anti-HCV shRNAs. (A) Design of shRNAs against HCV genome. Guide strand sequences (5′ to 3′) are listed. (B) On-target silencing efficacy and off-target effects were measured by dual-luciferase reporter assays in HEK293 cells as described earlier. *P (t-test, two tailed) < 0.0001 compared with sh-scramble control treatment. (C) The off-target spectrum of anti-HCV shRNAs was evaluated in vivo. shRNAs were expressed in mice and the resulting mRNA expression profiles in mouse livers were determined by RNA-Seq. All mRNAs with a value of FPKM higher than 2 were considered (n = 9000). Clustering of mRNA expression signature figures is shown. Average linkage hierarchical clustering was used with distance between samples measured by the square root of the Jensen–Shannon divergence (JSD). A low Jensen–Shannon distance corresponds to similar gene expression profiles (red shades of colors in the heat map). Scatter plots-based RNA expressions between individual samples and control can be found in Supplementary Figure S3H. Also see the Materials and Methods section for details.
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Figure 3: Silencing efficacy and off-targeting of anti-HCV shRNAs. (A) Design of shRNAs against HCV genome. Guide strand sequences (5′ to 3′) are listed. (B) On-target silencing efficacy and off-target effects were measured by dual-luciferase reporter assays in HEK293 cells as described earlier. *P (t-test, two tailed) < 0.0001 compared with sh-scramble control treatment. (C) The off-target spectrum of anti-HCV shRNAs was evaluated in vivo. shRNAs were expressed in mice and the resulting mRNA expression profiles in mouse livers were determined by RNA-Seq. All mRNAs with a value of FPKM higher than 2 were considered (n = 9000). Clustering of mRNA expression signature figures is shown. Average linkage hierarchical clustering was used with distance between samples measured by the square root of the Jensen–Shannon divergence (JSD). A low Jensen–Shannon distance corresponds to similar gene expression profiles (red shades of colors in the heat map). Scatter plots-based RNA expressions between individual samples and control can be found in Supplementary Figure S3H. Also see the Materials and Methods section for details.

Mentions: To validate our findings in a relevant preclinical setting, we created nine shRNAs against the hepatitis C virus (HCV) genome. All were designed as a 21-mer stem–loop structure with fixed ends as described previously, leaving only positions 2–18 as guide strand variables. Three were designed by the new scheme: GC-rich sequences in the central and AU-rich sequences in both seed and 3′ regions. Three shRNAs with balanced GC content (group I) and another three containing an AU-rich seed but overall balanced GC content (group II) were selected as controls (Figure 3A). As expected, all anti-HCV shRNAs had marginal passenger strand-mediated off-target effects (Supplementary Figure S3A) and relatively potent on-target activity (Figure 3B). In contrast, the guide-strand-mediated miRNA-like repression (off-targeting parameter) varied (Figure 3B). The degree of off-targeting did not correlate with the amount of guide strand (Supplementary Figure S3B), but rather with the GC content distribution profile. Off-targeting was observed in both control groups, but not with shRNAs containing AU-rich sequences in both seed and 3′ regions (Figure 3B). Similar observations were made in MEF cells (Supplementary Figure S3C). Together, our results demonstrate that potent anti-HCV shRNAs with reduced off-targeting could be achieved by following the new design scheme.


Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs.

Gu S, Zhang Y, Jin L, Huang Y, Zhang F, Bassik MC, Kampmann M, Kay MA - Nucleic Acids Res. (2014)

Silencing efficacy and off-targeting of anti-HCV shRNAs. (A) Design of shRNAs against HCV genome. Guide strand sequences (5′ to 3′) are listed. (B) On-target silencing efficacy and off-target effects were measured by dual-luciferase reporter assays in HEK293 cells as described earlier. *P (t-test, two tailed) < 0.0001 compared with sh-scramble control treatment. (C) The off-target spectrum of anti-HCV shRNAs was evaluated in vivo. shRNAs were expressed in mice and the resulting mRNA expression profiles in mouse livers were determined by RNA-Seq. All mRNAs with a value of FPKM higher than 2 were considered (n = 9000). Clustering of mRNA expression signature figures is shown. Average linkage hierarchical clustering was used with distance between samples measured by the square root of the Jensen–Shannon divergence (JSD). A low Jensen–Shannon distance corresponds to similar gene expression profiles (red shades of colors in the heat map). Scatter plots-based RNA expressions between individual samples and control can be found in Supplementary Figure S3H. Also see the Materials and Methods section for details.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231738&req=5

Figure 3: Silencing efficacy and off-targeting of anti-HCV shRNAs. (A) Design of shRNAs against HCV genome. Guide strand sequences (5′ to 3′) are listed. (B) On-target silencing efficacy and off-target effects were measured by dual-luciferase reporter assays in HEK293 cells as described earlier. *P (t-test, two tailed) < 0.0001 compared with sh-scramble control treatment. (C) The off-target spectrum of anti-HCV shRNAs was evaluated in vivo. shRNAs were expressed in mice and the resulting mRNA expression profiles in mouse livers were determined by RNA-Seq. All mRNAs with a value of FPKM higher than 2 were considered (n = 9000). Clustering of mRNA expression signature figures is shown. Average linkage hierarchical clustering was used with distance between samples measured by the square root of the Jensen–Shannon divergence (JSD). A low Jensen–Shannon distance corresponds to similar gene expression profiles (red shades of colors in the heat map). Scatter plots-based RNA expressions between individual samples and control can be found in Supplementary Figure S3H. Also see the Materials and Methods section for details.
Mentions: To validate our findings in a relevant preclinical setting, we created nine shRNAs against the hepatitis C virus (HCV) genome. All were designed as a 21-mer stem–loop structure with fixed ends as described previously, leaving only positions 2–18 as guide strand variables. Three were designed by the new scheme: GC-rich sequences in the central and AU-rich sequences in both seed and 3′ regions. Three shRNAs with balanced GC content (group I) and another three containing an AU-rich seed but overall balanced GC content (group II) were selected as controls (Figure 3A). As expected, all anti-HCV shRNAs had marginal passenger strand-mediated off-target effects (Supplementary Figure S3A) and relatively potent on-target activity (Figure 3B). In contrast, the guide-strand-mediated miRNA-like repression (off-targeting parameter) varied (Figure 3B). The degree of off-targeting did not correlate with the amount of guide strand (Supplementary Figure S3B), but rather with the GC content distribution profile. Off-targeting was observed in both control groups, but not with shRNAs containing AU-rich sequences in both seed and 3′ regions (Figure 3B). Similar observations were made in MEF cells (Supplementary Figure S3C). Together, our results demonstrate that potent anti-HCV shRNAs with reduced off-targeting could be achieved by following the new design scheme.

Bottom Line: The reduced off-targeting was confirmed by RNA-Seq analyses from mouse liver RNAs expressing various anti-HCV shRNAs.Compared with previously established work, the new algorithm was more effective in reducing off-targeting without jeopardizing on-target potency.These studies provide new rules that should significantly improve on siRNA/shRNA design.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.

Show MeSH
Related in: MedlinePlus