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. 2020 Mar 6;11(1):1223.
doi: 10.1038/s41467-020-15021-5.

Blackjack Mutations Improve the On-Target Activities of Increased Fidelity Variants of SpCas9 With 5'G-extended sgRNAs

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Free PMC article

Blackjack Mutations Improve the On-Target Activities of Increased Fidelity Variants of SpCas9 With 5'G-extended sgRNAs

Péter István Kulcsár et al. Nat Commun. .
Free PMC article

Abstract

Increased fidelity mutants of the SpCas9 nuclease constitute the most promising approach to mitigating its off-target effects. However, these variants are effective only in a restricted target space, and many of them are reported to work less efficiently when applied in clinically relevant, pre-assembled, ribonucleoprotein forms. The low tolerance to 5'-extended, 21G-sgRNAs contributes, to a great extent, to their decreased performance. Here, we report the generation of Blackjack SpCas9 variant that shows increased fidelity yet remain effective with 21G-sgRNAs. Introducing Blackjack mutations into previously reported increased fidelity variants make them effective with 21G-sgRNAs and increases their fidelity. Two "Blackjack" nucleases, eSpCas9-plus and SpCas9-HF1-plus are superior variants of eSpCas9 and SpCas9-HF1, respectively, possessing matching on-target activity and fidelity but retaining activity with 21G-sgRNAs. They facilitate the use of existing pooled sgRNA libraries with higher specificity and show similar activities whether delivered as plasmids or as pre-assembled ribonucleoproteins.

Conflict of interest statement

Biospirál-2006 Ltd has filed a patent (Application number 128977–18457) concerning the use of Blackjack variants described in this manuscript with P.I.K., A.T., E.T., A.N., Z.L., Z.W. and E.W. as listed inventors.

Figures

Fig. 1
Fig. 1. Structure-guided mutagenesis increases on-target activity of SpCas9-HF1 with 21G-sgRNAs.
a X-ray crystallography derived structure of SpCas9-sgRNA-DNA complex in the conformation closest to the cleavage-competent state (PDB ID: 5f9r). b Sequences of SpCas9-HF1 and the selected Blackjack-SpCas9-HF1 at the region affected, between residues L1004 and D1017; deletions (−) and insertions (green) are indicated. See also Supplementary Fig. 1. c, d Blackjack mutations increase on-target activities of increased fidelity variants with 21G-sgRNAs on different targets. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).
Fig. 2
Fig. 2. The Blackjack mutations increase not only the activity of increased fidelity nucleases charged with 21G-sgRNAs, but their target-selectivity in general.
a Blackjack mutations increase the target-selectivity of their respective parent SpCas9 variants. EGFP-disruption activities with perfectly matching 20G-sgRNAs. Results are shown only for those target sites where the SpCas9 variant without Blackjack mutations exhibits higher than background level cleavage. See also Supplementary Fig. 2. b On-target activities with 21G-sgRNAs on more target sites for which the SpCas9 variant with Blackjack mutations using 20G-sgRNAs exhibits at least 70% on-target activity compared to WT SpCas9. No target corresponds to this condition in the case of HeFSpCas9. See also Supplementary Fig. 3. a, b The median and the interquartile range are shown; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter and an appended 21st nucleotide by a red lowercase letter. Statistical significance was assessed using two-sided Paired-samples Student’s t-test or two-sided Wilcoxon signed ranks test as appropriate; ns not significant. A summary of data distributions and statistical details is reported in Supplementary Data 6.
Fig. 3
Fig. 3. The Blackjack mutations increase the fidelity of increased fidelity nucleases.
a Blackjack mutations increase the fidelity of their respective parent SpCas9 variants. EGFP-disruption activities with partially mismatching 20G-sgRNAs. Results are shown only for those target sites where both the non-Blackjack parent- and Blackjack-SpCas9 variant exhibit at least 70% on-target activity (with perfectly matching 20G-sgRNAs) compared to WT SpCas9. Only one target (with three mismatched positions) matches this condition in the case of evo- or HeFSpCas9. The median and the interquartile range are shown; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of a mismatching nucleotide (not necessarily the exact position) within the spacer; numbering of the tooth positions corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter. Statistical significance was assessed using two-sided Paired-samples Student’s t-test or two-sided Wilcoxon signed ranks test as appropriate; ns not significant. Summary of data distributions and statistical details are reported in Supplementary Data 6. See also Supplementary Fig. 4. b Bar chart of the total number of off-target sites detected by GUIDE-seq for WT and B-SpCas9 variants on six target sites targeted with 20G- or 21G-sgRNAs. See also Supplementary Fig. 5.
Fig. 4
Fig. 4. Restoring mutations to WT amino acids lowers the (on-)target-selectivity and fidelity of B-eSpCas9 and B-SpCas9-HF1.
a, d Schematic representation of the mutations in each variant examined. b, e On-target activities using 20G-sgRNAs measured on five target sites (n = 3 biologically independent samples [overlaid as blue circles]), employing the EGFP-disruption assay, median and interquartile range are shown. c, f Mismatch screen results from EGFP-disruption assay. Target sites and matching (e.g., T1, T6) or mismatching sgRNAs (e.g., T1MM1, T6MM1) are the same as in Supplementary Fig. 3. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of a mismatching nucleotide (not necessarily the exact position) within the spacer; numbering of the tooth positions corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).
Fig. 5
Fig. 5. eSpCas9-plus and SpCas9-HF1-plus show greatly enhanced on-target activity with 21G-sgRNAs and identical fidelity/target-selectivity compared to eSpCas9 and SpCas9-HF1, respectively, as assessed by EGFP disruption, indels measured by NGS and by GUIDE-seq.
ac EGFP-disruption activity a with 20G-sgRNAs targeting 25 sites; b, c with either 20G- or 21G-sgRNA pairs targeting two alternative sets of 10 different sequences shown as the ratio of variant activity to WT activity. d, e On-target activities of SpCas9 variants across 23 endogenous target sites within the human VEGFA or FANCF loci targeted with d 20G- or e 21G-sgRNAs, measured by amplicon resequencing. f Bar chart of the total number of off-target sites detected by GUIDE-seq for SpCas9 variants on seven sites targeted with 20G-sgRNAs. ae Tukey-type boxplots by BoxPlotR: center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the “minimum” and “maximum” data situated within 1.5 times the interquartile range from the 25th and 75th percentiles, respectively; notches indicate the 95% confidence intervals for the medians; crosses represent sample means; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter and an appended 21st nucleotide by a red lowercase letter. See also Supplementary Figs. 6 and 7.
Fig. 6
Fig. 6. The plus variants are effective when transfected in pre-assembled RNP form.
ac EGFP-disruption assays. Target sequences start with 5′ non-G-, G- or GG-nucleotides. Data from the individual samples are detailed in Supplementary Fig. 8c–e. Tukey-type boxplots by BoxPlotR: center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the “minimum” and “maximum” data situated within 1.5 times the interquartile range from the 25th and 75th percentiles, respectively; notches indicate the 95% confidence intervals for the medians; crosses represent sample means; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide or dinucleotide of the spacer is indicated by an uppercase letter and an appended 21st and 22nd nucleotides by red lowercase letters. See also Supplementary Fig. 8.
Fig. 7
Fig. 7. Blackjack variants facilitate modification of the endogenous Sprn gene at the 5′ coding region and are effective with sgRNAs expressed from a single-copy lentivirus.
a Pre-screening targets with increased fidelity nucleases for efficiency by the integration of a donor EGFP cassette. b Based on a the SpCas9-plus variants were selected to generate transgenic lines using the ‘self-cleaving’ EGFP-expression plasmid, which must integrate in-frame for Sprn promoter driven EGFP expression, and which downstream from the EGFP coding sequence it also contains a CMV-mCherry cassette; mCherry positive cells were counted. c Indel formation activity of eSpCas9-plus compared to WT and eSpCas9 with 21G-sgRNAs transcribed from an integrated single copy of lentiviruses measured by TIDE. ac Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles). In the case of VEGFA site 8 targeted with WT and eSpCas9-plus on c one sample point is missing due to sample loss. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter and an appended 21st nucleotide by a red lowercase letter.

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References

    1. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–821. doi: 10.1126/science.1225829. - DOI - PMC - PubMed
    1. Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nat. Methods. 2013;10:957–963. doi: 10.1038/nmeth.2649. - DOI - PMC - PubMed
    1. Jinek M, et al. RNA-programmed genome editing in human cells. elife. 2013;2:e00471. doi: 10.7554/eLife.00471. - DOI - PMC - PubMed
    1. Mali P, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–826. doi: 10.1126/science.1232033. - DOI - PMC - PubMed
    1. Cong L, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–823. doi: 10.1126/science.1231143. - DOI - PMC - PubMed
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