CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs

Abstract

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.

Fig. 1. CRISPR GUARD protection of Cas9 off-target sites.

Schematic showing how short gRNAs (orange) forming catalytically inactive complexes with Cas9 can occupy specific off-target sites in the genome and compete with the mismatched nuclease competent gRNA (black), thereby providing protection from Cas9-mediated DNA cleavage. Mismatches in the gRNA are shown in red. RNA bases overlapping the gRNA PAM are shown in bold.

Fig. 2. GUARD RNAs reduce Cas9 off-target cleavage activity without affecting on-target editing.

a Bio-layer interferometry (BLI) analysis of binding kinetics for dead Cas9 ribonucleoprotein complex to an immobilised biotinylated DNA substrate. Cas9 was precomplexed with tracrRNA and (NT) non-targeting control gRNA; (gRNA) 20-nt VEGFA gRNA with 4 mismatches; (GUARD RNA) a 15-nt GUARD RNA targeting the off-target site or no guide RNA. b CRISPR GUARD design. Schematic showing GUARD RNAs designs. Variation in length (14-nt versus 15-nt) and protospacer positioning likely influence binding energy and competition with the on-target gRNA (i.e., competitive/overlapping, non-competitive/proximal). Mismatches in the gRNA are shown in red. RNA bases overlapping the gRNA PAM are shown in bold. c Competition between on-target gRNA and GUARD RNA can be reduced by proximal positioning. On-target (VEGFA) and off-target (CAVIN4) DNA abundance was measured by qPCR following an in vitro Cas9 DNA cleavage assay. The corresponding GUARD RNA was added at a molar ratio of 5:1 to the VEGFA on-target gRNA (100 nM to 20 nM). d GUARD RNAs are more effective at blocking Cas9-mediated DNA cutting with pre-incubation in vitro. On-target (VEGFA) and off-target (CAVIN4) DNA abundance was measured by qPCR following an in vitro Cas9 DNA cleavage assay, with a 30 min pre-incubation with GUARD RNAs and Cas9, before addition of the on-target gRNA and Cas9. Increasing molar ratios of GUARD RNA to the VEGFA on-target gRNA were used (10:10, 20:10, 50:10, 100:10 nM). e GUARD RNAs are effective in blocking Cas9-mediated DNA cleavage of off-target sites within a 10 bp window of the gRNA PAM. Synthetic DNA fragments containing the VEGFA gRNA off-target site CAVIN4 positioned progressively further away from the GUARD RNA binding site were quantified by qPCR following an in vitro Cas9 DNA cleavage assay (50:10 nM GUARD RNA to gRNA ratio). f CRISPR GUARD is effective at blocking off-target editing in cells. Indel rates from NGS of amplicons from Cas9-expressing HEK293 cells transfected with VEGFA gRNA and multiple GUARD RNA designs for protection of the CAVIN4 proximal off-target site (25:25 nM GUARD RNA to gRNA ratio). Data represent the mean of two (f) independent experiments, or the mean ± SD of three (c, e) or five (d) independent experiments with symbols representing each replicate. For a, data are representative of two independent experiments. NT non-targeting gRNA, UTC untransfected control. Unpaired, two-tailed student’s t-test. For c, *P = 0.0208, **P = 0.0083. For d, *P = 0.0164, **P = 0.0021 (VEGFA) and **P = 0.007 (CAVIN4), ***P = 0.0003, ***P < 0.0001. For e, **P = 0.0041 and ***P = 0.0001. Source data are provided as a Source Data file.

Fig. 3. Protection of multiple, endogenous off-target sites with CRISPR GUARD.

a Increasing GUARD RNA concentration leads to complete protection of Cas9 off-target sites in cells. Indel rates from NGS of amplicons from Cas9-expressing HEK293 cells transfected with VEGFA gRNA and increasing concentrations of 14-nt GUARD RNA for protection of the CAVIN4 off-target site (10:10, 20:10, 50:10, 100:10 nM). The total concentration of delivered RNA in each case was kept constant by co-delivery of NT gRNA (first condition is 100 nM NT). b Indel rates from amplicons sequencing of Cas9-expressing HEK293 cells transfected with EMX1 or VEGFA on-target gRNAs, and a single GUARD RNA (25:25 nM ratio). GUARD RNA positioning relative to the off-target protospacer sequence is shown schematically in each panel. Data represent the mean of two independent experiments with symbols representing each replicate. Source data are provided as a Source Data file.

Fig. 4. GUARD RNAs can reduce off-target base editing if designed to avoid cytosine exposure.

a Protection from off-target cytosine deamination with CRISPR GUARD. Base editing rates from amplicon sequencing of BE3-expressing HEK293 cells transfected with EMX1 or VEGFA on-target gRNA (25 nM) and the indicated GUARD RNA (125 nM). Shown is the cumulative editing rate for the cytosines highlighted. The predicted cytosine deamination activity window is indicated. b Increased base editing frequencies with GUARD RNAs that expose cytosines. Base editing rates from amplicon sequencing of BE3-expressing HEK293 cells transfected with EMX1 (left) or VEGFA (right) on-target gRNA (25 nM) and the indicated GUARD RNA (125 nM). c GUARD RNAs can facilitate base editing without full-length on-target gRNAs. Base editing rates from amplicon sequencing of BE3-expressing HEK293 cells transfected with MFAP1 GUARD RNA only (125 nM). Data represent the mean of two independent experiments with symbols representing each replicate. Source data are provided as a Source Data file.

Fig. 5. High-throughput screening identifies functional GUARD RNAs.

a Schematic of the lentiviral pooled screening approach to analyse off-target editing frequencies in cells expressing different GUARD RNA species. Numbers indicate the length of the plasmid segment in base pairs. b Scatter plots of indels (Cas9) or SNPs (base edits) for two independent replicate experiments with or without induction of Cas9 or BE3 expression with doxycycline for 48 h. c Box and whiskers plot of off-target indel or base editing frequency for coding and non-targeting (NT) GUARD RNAs in the Cas9 or base editor screen, respectively. NB: some outliers fall outside of the y-axis limit. d GUARD RNAs binding to the same DNA strand as the gRNA are more effective at blocking Cas9 off-target editing but can increase base editing efficiency. Box and whiskers plot of off-target indel or SNP frequencies in the screens. NB: some outliers fall outside of the y-axis limit. Box and whiskers plot: centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. Data were compared using unpaired, two-tailed student’s t-tests.

Fig. 6. GUARD RNA features that significantly reduce off-target editing.

a GUARD RNA performance for Cas9 and, (b) BE3. Base editing rates for BE (% of NGS reads with SNPs) or indel rates for Cas9 (% of NGS reads with indels) for a subset of off-target sites. Off-targets are labelled with an identifier, gene name (if genic), gRNA name, and the number of mismatches between the gRNA and the off-target site. Data are pooled from two independent experiments from cells treated with doxycycline. Each point represents a GUARD RNA and are compared to three non-targeting (NT) control RNAs. GUARD RNAs are classed as functional if they reduce off-target editing by at least two standard deviations from the mean of the NT controls. Data shown here is a subset of that presented in Supplementary Fig. 10. c Lentiviral plasmid screening reveals GUARD RNAs that are effective at endogenous genomic loci. (Left panel) indel rates from GUARD RNA screening of EMX1 gRNA off-target site MFAP1, highlighting non-functional GUARD RNA (v1) and functional GUARD RNA (v2). Screening data shown here is a subset of that presented in Supplementary Fig. 10. (Right panels) indel rates from amplicon sequencing of on-target and off-target endogenous loci from Cas9-expressing HEK293 cells transfected with EMX1 gRNA and GUARD RNA v1, GUARD RNA v2 or a NT control (all at 25 nM). Data represent mean of two independent experiments with symbols representing each replicate. d GUARD RNAs can mediate base editing at position 1 and 2 within the 15-nt GUARD RNA. GUARD RNAs were grouped according to whether they had a cytosine at each nucleotide position or not, and P values were generated by comparing the % of NGS reads with SNPs between GUARD RNA groups with an unpaired, two-tailed student’s t-tests. e Functional GUARD RNAs predominantly have NGG PAMs. The proportion of functional GUARD RNAs from the Cas9 screen that had NGG PAMs versus NAG PAMs was compared using a two-sided Fisher’s exact test. f Model depicting important parameters for GUARD RNA design. For GUARD RNAs that reduce Cas9 off-target editing, reducing distance from the gRNA, having an NGG PAM, binding to the same DNA strand as the gRNA, increased GC-content and high concentration in the cell will all increase the effectiveness of CRISPR GUARD. For BE3, the presence of cytosines at position 1 or 2 is to be avoided for 15-nt GUARD RNAs to prevent editing. Source data are provided as a Source Data file.