Moreno-Mateos MA et al. (2015), CRISPRscan: designing highly efficient sgRNAs f...

XB-ART-51392

Nat Methods 2015 Oct 01;1210:982-8. doi: 10.1038/nmeth.3543.

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CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.

Moreno-Mateos MA , Vejnar CE , Beaudoin JD , Fernandez JP , Mis EK , Khokha MK , Giraldez AJ .

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CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5' mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3' UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.

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Species referenced: Xenopus
Genes referenced: dicer1 gnl3 ndrg1 s1pr2 tbx6 tubg1

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	Figure 2. Stable sgRNAs are more active, G-rich and A-depleteda. Biplot of sgRNA levels (log2 RPM) comparing 0 and 1.25 hpf (Experiment shown in Figure 1), colored to indicate the frequencies of the 4 nucleotides in each sgRNA. Corresponding Spearman correlations between nucleotide frequencies and sgRNA stability (ratio of 1.25 hpf to 0 hpf levels) are shown (right), with p values indicated.b. Biplot illustrating stable and unstable groups of sgRNAs, defined by >2-fold enrichment or depletion between 0 and 1.25 hpf (log2 RPM). sgRNAs with low read-counts (bottom 10%) were excluded (grey lines).c. Barplot representing the nucleotide composition of the 20% most stable sgRNAs compared to all others. Bars show log-odds scores of nucleotide frequencies for each position in the sgRNA (1 to 20) (G-test: * <0.05, <0.01).d. Box and whisker plots (Box spans first to last quartiles with 1.5x interquartile range distance for whiskers) showing sgRNA activity (left) and the input levels (right) in the stable and unstable sgRNAs. Mann-Whitney U test (** p< 0.0001, ns: not significant).e. Diagram illustrating the experiment to analyze the Cas9 loading activity in vivo. One-cell stage embryos were injected with 1280 sgRNAs in pools of 80 along with FLAG-cas9 mRNA (see Methods). sgRNA levels were measured at 0 and 1.25 hpf and immunoprecipitation of FLAG-Cas9 was performed at 6 and 9 hpf to analyze levels of loaded sgRNAs. Analysis of the immunoprecipitation of FLAG-Cas9 at 6 hpf (bottom). 1/50 of the total input (IN), the immunoprecipitation (IP) and the supernatant after the immunoprecipitation (SN) were analyzed by western blot using FLAG and gamma-tubulin (as loading control) antibodies.f. Biplot of sgRNA levels (log2 RPM) comparing 1.25 hpf and loaded into Cas9 at 6 hpf, colored to indicate the frequencies of A and G in each sgRNA. Corresponding Spearman correlations between nucleotide frequencies and sgRNA stability (ratio of loaded at 6h to 1.25 hpf levels) are shown (right) with p values indicated.g. Biplot of sgRNA levels comparing 0 hpf and loaded into Cas9 at 6 hpf. (see panel f).
	Figure 3. CRISPR/Cas9 activity is modulated by the sgRNA sequencea. Barplot representing the nucleotide composition of the 20% most efficient sgRNA sites (positions 1 to 20 extended by the PAM sequence and 6 nt) compared to all others. Bars show log-odds scores of nucleotide frequencies for each position (G-test: * <0.05, ** <0.01).b. Performance of linear regression based prediction model (CRISPRscan). sgRNAs were divided into quintiles based on CRISPRscan scores (horizontal axis), then each quintile was evaluated based on their experimentally determined activities (colors indicate five activity levels).c. Diagram showing 11 sgRNA sites targeting albino exons 1 and 2 used in an independent validation of the prediction model. Phenotypes obtained after the injection of the sgRNAs, showing different levels of mosaicism compared to the wild type (WT) (bottom). Lateral views and insets of the eyes of 48 hpf embryos are shown. Picture of an albino loss of function mutant (â/â) described in White et al.17 (right). (scale bars: 1mm, 0.25mm inset)d. Phenotypic evaluation of 11 sgRNA targeting albino. Stacked barplots showing the percentage of albino like (white), mosaic (gray) and phenotypically WT (black) embryos 48 hpf after injection. Predicted CRISPRscan scores, ranks and number of embryos evaluated (n) are shown for each sgRNA.e. Scatter plot showing the correlation between CRISPRscan scores and experimentally measured activities based on all phenotypes used to independently validate CRISPRscan (Panel c and d and Supplementary Fig. 4). Spearman correlation and p value are indicated.
	Figure 4. Extending the CRISPR target repertoire with truncated, extended and 5â² mismatch-containing sgRNAsa. Boxplot representing the ranked normalized activity of each class of alternative sgRNAs, ordered by median activity. Shorter sgRNAs (GG16 and GG17) and sgRNAs inducing 1 mismatch in the 5â² GG (gG18 and Gg18) are the most active alternatives to the canonical GG18 sgRNA.b. Biplot of sgRNA levels (log2 RPM) comparing 0 and 1.25 hpf, colored to indicate the frequencies of A and G in each sgRNA. Corresponding Spearman correlations between nucleotide frequencies and sgRNA stability (ratio of 1.25 hpf to 0 hpf levels) are shown (right), with p values indicated.c. Biplot illustrating stable and unstable groups of sgRNAs, defined by >2-fold enrichment or depletion between 0 and 1.25 hpf (log2 RPM) (left). sgRNAs with low read-counts (bottom 10%) were excluded (grey lines). Box and whisker plots showing sgRNA activity (middle) and the input levels (right) in the stable and unstable sgRNAs. Mann-Whitney U test (**** p< 0.0001, ns: not significant).
	Figure 5. Targeting CRISPR/Cas9 activity to germ cellsa. Wild-type embryos were injected with a combination of 3 sgRNAs (20 pg each) targeting ntla, tbx6 or ndr1/2 and 100 pg of cas9 mRNA (150 in the case of ndr1/2). Pictures were taken at 28 hpf (lateral view). Arrows are indicating cyclopia (ndr1/2) and fused somites (tbx6). (scale bar: 0.5mm)b. Schema illustrating the Cas9-nanos 3â²-UTR strategy. Nanos 3â²-UTR was cloned after Cas9 ORF. Injection of Cas9-nanos will concentrate the expression in the germ cells (green circles).c. Stacked barplot showing the percentage of coherent F0 phenotype (mutant phenotype) or WT after injection with a combination of 3 sgRNAs (20 pg each) targeting s1pr2 or ntla and using cas9-globin or cas9-nanos mRNA (100 pg). n= number of embryos analyzed. Ï2 test (**** p< 0.0001).d. Individual pictures (lateral view) of 48 hpf old embryos injected with the same sgRNA/Cas9 combinations described in panel c (targeting ntla). (scale bar: 1mm)e. Survival curve of Cas9-nanos or Cas9-globin injected fishes. Ï2 test (* < 0.05) Embryos were injected with the same sgRNA/Cas9 combinations described in panel c (targeting dicer1).f. Pictures showing fish injected with sgRNA (dicer1) and Cas9-nanos or Cas9-globin 4 months of age. Box and whisker plot showing their size distribution (right). n= number of embryos analyzed. Two-tailed Studentâs t test (**** p< 0.0001).g. Scheme illustrating an F0 out cross between sgRNA (dicer1), Cas9-nanos or Cas9-globin injected female fish and dicer1 â/â mutants males generated by germ line transplantation24. Pictures of 32 hpf MZ dicer1 embryos derived from F0 females injected with Cas9-nanos and sgRNA (dicer1) (right). (scale bar: 0.5mm)

References [+] :

Bassett, CRISPR/Cas9 and genome editing in Drosophila. 2014, Pubmed