Rescue of high-specificity Cas9 variants using sgRNAs with matched 5' nucleotides

Abstract

We report that engineered Cas9 variants with improved specificity-eCas9-1.1 and Cas9-HF1-are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5' terminus, relative to target DNA sequences. Because the nucleotide at the 5' end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5' nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity.

Keywords: CRISPR-Cas; Engineered Cas9 variants; Hammerhead ribozyme-linked sgRNA; Off-target effect.

Fig. 1

High-specificity Cas9 variants with attenuated cleavage activity. a Schematics of Cas9-WT, engineered Cas9 variants, and sgRNA variants. Locations of the alanine substitutions introduced in Cas9-WT to create eCas9-1.1 or Cas9-HF1 are shown with blue or red asterisks, respectively. Red triangles indicate cleavage positions. GX₁₉ sgRNA starts with a G matched to its protospacer (blue line). gX₁₉ sgRNA has a mismatched G at its 5’-end. gX₂₀ sgRNA includes an extra guanine at its 5’ terminus. The protospacer-adjacent motif (PAM) is shown by a red line labeled NGG. H, not G (A or C or T); D, not C (A or G or T). b, c Western blot (b) and indel frequencies at the EMX1 site (c) of HeLa cells co-transfected with plasmids encoding Cas9-WT or variants and EMX1-specific sgRNA. Cas9 variants were expressed by new plasmids used in this study or by old plasmids used in previous studies. Error bars, s.e.m of two or three biological replicates

Fig. 2

Reduced editing activity of high-fidelity Cas9 variants at target sites with a non-G 5’ nucleotide. a Indel frequencies of Cas9-WT and Cas9 variants at 26 endogenous target sites with an HX₁₉ sequence obtained using gX₁₉ sgRNAs in HeLa cells (H: not G [C or T or A]). The sequences and indel frequencies (%) of the 26 target sites are described in Additional file 1: Table S1. * P < 0.05, ** P < 0.01, **** P < 0.0001. b Distributions of relative indel frequencies of eCas9-1.1 and Cas9-HF1 normalized to that of Cas9-WT. Means and medians of relative indel frequencies are represented in Additional file 1: Table S2. Box and whisker plots: center lines show the medians; crosses show the means. c Indel frequencies (measured by targeted deep sequencing) at six on-target sites with a 5’ C or 5’ T nucleotide obtained using gX₁₉ and gX₂₀ sgRNAs in HeLa cells. PAM sequences are shown in blue. Error bars, s.e.m. of three biological replicates

Fig. 3

Recovery of editing efficiency of high-fidelity Cas9 variants using HH ribozyme-linked sgRNAs. a A schematic of a self-processing ribozyme fused sgRNA. The pre-sgRNA contains a HH ribozyme at its 5’-end. The pre-sgRNA undergoes self-cleavage to release a mature sgRNA. The red arrow indicates the self-cleavage position. b HH ribozyme-fused sgRNAs with a matched 5’ nucleotide (HH-X₂₀) or a mismatched guanosine (HH-gX₁₉) were tested in combination with Cas9-WT and high-fidelity Cas9 variants at six target sites in HeLa cells. Indel frequencies were measured using targeted deep sequencing. The PAM is shown in blue. Error bars, s.e.m. of three biological replicates. Statistical significances were calculated by unpaired t-test. * P < 0.05, ** P < 0.01. c Mean indel frequencies ± s.e.m. at the six target sites in HeLa cells. Statistical significances were calculated by paired t-test. ** P < 0.01, *** P < 0.001

Fig. 4

Specificities of high-fidelity Cas9 variants in combination with HH-X₂₀ sgRNA. The HH-X₂₀ sgRNA expression plasmid was co-transfected with the plasmid encoding Cas9-WT or a Cas9 variant into HeLa cells. Indel frequencies at on- and off-target sites were measured by targeted deep sequencing. PAM sequences are shown in blue. Mismatched bases are shown in red. The specificity ratio indicates the fold difference between the ratio of on-target indel frequencies to off-target indel frequencies obtained using Cas9 variants and that obtained using Cas9-WT. Error bars, s.e.m. of three biological replicates. Indel frequencies significantly above those of the mock transfected sample are shown by asterisks (* P < 0.05, ** P < 0.01)