Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

Abstract

Background: The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects.

Results: These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5' G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s.

Conclusions: No single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high-fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple means for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.

Keywords: CRISPR; Cas9; Disruption assay; Extended sgRNA; HeFSpCas9; HeFm2SpCas9; High fidelity nuclease; Off-target; SpCas9-HF1; Truncated sgRNA; eSpCas9; sgRNA.

Fig. 1

SpCas9 variants employed in these studies. Schematics depicting the main features of the wild type and the five mutant variants of SpCas9 used: each protein sequence is flanked by a nuclear localization signal (NLS) at both ends and is preceded by a 3xFLAG tag. HeF-variants containing combinations of mutations from both eSpCas9 and SpCas9-HF1

Fig. 2

Extending the guide RNA with a matching 5′ end G nucleotide is much more detrimental their activities than extending with a mismatching one in the case of eSpCas9 and SpCas9-HF1. Effect of 5′ extension of the sgRNA with a a mismatching G or b a matching G nucleotide on the activities of SpCas9 nucleases in comparison with using perfectly matching 20-nucleotide-long spacers (data used are from Additional file 1: Figure S2a, c and S2b, d; sites targeted are provided in Additional file 2). Schematics for the spacers used are depicted below the categories as green combs and the 21st G nucleotide extensions are depicted as a red bent end tooth if mismatching; lower case g represents appended nucleotides; numbering corresponds to the distance from the PAM. Tukey-type notched boxplots by BoxPlotR [67]: center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; notches indicate the 95% confidence intervals for medians; crosses represent sample means; data points are plotted as open circles and correspond to the different targets tested (in total 26 and 10, respectively, for a and b). Each pair of means is statistically different at the p < 0.05 level: a SpCas9–eSpCas9 (<0.001), SpCas9–SpCas9-HF1 (<0.001), SpCas9-HF1–eSpCas9 (<0.015); b statistically different pairs SpCas9–eSpCas9 (<0.001), SpCas9–SpCas9-HF1 (<0.001)

Fig. 3

Side-by-side comparison of SpCas9 variants programmed with perfectly matching sgRNAs reveal a target-selectivity ranking among the variants in the order of eSpCas9 > SpCas9-HF1 > HeFSpCas9. a EGFP disruption activities of the nucleases, calculated as described in “Methods”. Bars correspond to averages of n = 3 parallel samples; error bars represent the standard errors estimated by Gaussian error propagation of the component standard deviations associated with both EGFP and mCherry (transfection control) values. The target numbers in squares are the 16 targets examined in Fig. 4a. b Summary of the characteristics of distributions of data for on-target disruption activities of nuclease variants normalized to that of the WT SpCas9. Tukey-type notched boxplots by BoxPlotR: center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; notches indicate the 95% confidence intervals for medians; crosses represent sample means; data points are plotted as open circles. The sample points (24 in case of each variant) correspond to the targets present on Fig. 3a. Each pair of means is statistically different at the p < 0.05 level: SpCas9-HF1–eSpCas9 (<0.002), SpCas9-HF1–HeFSpCas9 (<0.001), HeFSpCas9–eSpCas9 (<0.001)

Fig. 4

Cleavability ranking of the targets by nuclease variant as well as fidelity ranking (eSpCas9 < SpCas9-HF1 < HeFSpCas9) of the nucleases on these targets is apparent. Disruption and indel formation activities of SpCas9 nucleases programmed with perfectly matching or partially mismatching sgRNAs. a Heat maps showing the relative activities (white to green) of the nuclease variants compared to the WT for each of the targets and the ratios of off-target to on-target disruption activities (blue to white) of the WT and mutant nucleases measured employing the indicated target and mismatching spacer sequences; grey and black boxes indicate not determined due to diminished on-target activities and sample loss, respectively. b–d Specificities (on-target:off-target ratio) of the nucleases assessed by b disruption activities, c deep-sequencing on indel formation (eSpCas9 and SpCas9-HF1) and disruption activities (SpCas9), and d deep-sequencing on indel formation (HeFSpCas9) and disruption activities (SpCas9, eSpCas9, SpCas9-HF1)

Fig. 5

Variant HeFm2SpCas9 exhibits target selectivity and fidelity between those of SpCas9-HF1 and HeFSpCas9. Disruption and indel formation activities of SpCas9 nuclease variants bearing combinations of eSpCas9 and SpCas9-HF1 mutations. a Tukey-type notched boxplots of ratios of on-target disruption activities of the variants to those of WT nucleases as indicated (data used are from Additional file 1: Figure S9a, b; sites targeted are provided in Additional file 2): center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; notches indicate the 95% confidence intervals for medians; crosses represent sample means; data points are plotted as open circles (17 in case of each variant) and correspond to the targets tested. Statistically different pairs of means at the p < 0.05 level: SpCas9HF1–HeFm1SpCas9 (<0.001), SpCas9HF1–HeFm2SpCas9 (<0.015), SpCas9HF1–HeFSpCas9 (<0.001). b Comparison of specificities of SpCas9-HF1 and HeFm2SpCas9 assessed in disruption assays with partially mismatching sgRNAs on targets where HeFm2SpCas9 reached at least 60% activity of the WT protein as in a. c Left panel: mean percentage modifications by WT SpCas9 and variants at FANCF site 2 as well as off-target site 1 from Fig. 5 in [33], which is readily cleaved by SpCas9-HF1. Percentage modifications were determined by TIDE; error bars represent standard deviation with Gaussian error propagation for n = 3 parallels. Right panel: specificity of WT and mutant variants on the FANCF site 2 plotted as ratio of on-target to off-target activity (calculated from the left panel)

Fig. 6

Transcription activation is not impaired compared to the WT when the nuclease variants are charged with altered sgRNAs. Transcription activation of an EGFP inserted after the Prnp promoter, employing five targets in the promoter region. a Active nuclease variants programmed with 15-nucleotide-long truncated MS2 aptamer-containing sgRNAs. b Dead nuclease variants programmed with 21-nucleotide-long MS2 aptamer-containing sgRNAs. Spacers used are shown as combs where green represents matching and red mismatching positions; lower case g represents appended nucleotides to the 5′ end of the guide; numbering corresponds to the distance from the PAM. Bars correspond to averages of n = 3 parallel samples; error bars represent the standard deviations