RNA editing with CRISPR-Cas13

Abstract

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.

Figure 1. Characterization of a highly active Cas13b ortholog for RNA knockdown

A) Schematic of stereotypical Cas13 loci and corresponding crRNA structure. B) Evaluation of 19 Cas13a, 15 Cas13b, and 7 Cas13c orthologs for luciferase knockdown using two different guides. Orthologs with efficient knockdown using both guides are labeled with their host organism name. Values are normalized to a non-targeting guide with designed against the E. coli LacZ transcript, with no homology to the human transcriptome. C) PspCas13b and LwaCas13a knockdown activity (as measured by luciferase activity) using tiling guides against Gluc. Values represent mean +/− S.E.M. Non-targeting guide is the same as in Fig. 1B. D) PspCas13b and LwaCas13a knockdown activity (as measured by luciferase activity) using tiling guides against Cluc. Values represent mean +/− S.E.M. Non-targeting guide is the same as in Fig. 1B. E) Expression levels in log₂(transcripts per million (TPM+1)) values of all genes detected in RNA-seq libraries of non-targeting control (x-axis) compared to Gluc-targeting condition (y-axis) for LwaCas13a (red) and shRNA (black). Shown is the mean of three biological replicates. The Gluc transcript data point is labeled. Non-targeting guide is the same as in Fig1B. F) Expression levels in log₂(transcripts per million (TPM+1)) values of all genes detected in RNA-seq libraries of non-targeting control (x-axis) compared to Gluc-targeting condition (y-axis) for PspCas13b (blue) and shRNA (black). Shown is the mean of three biological replicates. The Gluc transcript data point is labeled. Non-targeting guide is the same as in Fig. 1B. G) Number of significant off-targets from Gluc knockdown for LwaCas13a, PspCas13b, and shRNA from the transcriptome wide analysis in E and F.

Figure 2. Engineering dCas13b-ADAR fusions for RNA editing

A) Schematic of RNA editing by dCas13b-ADAR_DD fusion proteins. Catalytically dead Cas13b (dCas13b) is fused to the deaminase domain of human ADAR (ADAR_DD), which naturally deaminates adenosines to insosines in dsRNA. The crRNA specifies the target site by hybridizing to the bases surrounding the target adenosine, creating a dsRNA structure for editing, and recruiting the dCas13b-ADAR_DD fusion. A mismatched cytidine in the crRNA opposite the target adenosine enhances the editing reaction, promoting target adenosine deamination to inosine, a base that functionally mimics guanosine in many cellular reactions. B) Schematic of Cypridina luciferase W85X target and targeting guide design. Deamination of the target adenosine restores the stop codon to the wildtype tryptophan. Spacer length is the region of the guide that contains homology to the target sequence. Mismatch distance is the number of bases between the 3’ end of the spacer and the mismatched cytidine. The cytidine mismatched base is included as part of the mismatch distance calculation. C) Quantification of luciferase activity restoration for dCas13b-ADAR1_DD(E1008Q) (left) and dCas13b-ADAR2_DD(E488Q) (right) with tiling guides of length 30, 50, 70, or 84 nt. All guides with even mismatch distances are tested for each guide length. Values are background subtracted relative to a 30nt non-targeting guide that is randomized with no sequence homology to the human transcriptome. D) Schematic of the sequencing window in which A to I edits were assessed for Cypridinia luciferase W85X. E) Sequencing quantification of A to I editing for 50-nt guides targeting Cypridinia luciferase W85X. Blue triangle indicates the targeted adenosine. For each guide, the region of duplex RNA is outlined in red. Values represent mean +/− S.E.M. Non-targeting guide is the same as in Fig. 2C.

Figure 3. Measuring sequence flexibility for RNA editing by REPAIRv1

A) Schematic of screen for determining Protospacer Flanking Site (PFS) preferences of RNA editing by REPAIRv1. A randomized PFS sequence is cloned 5’ to a target site for REPAIR editing. Following exposure to REPAIR, deep sequencing of reverse transcribed RNA from the target site and PFS is used to associate edited reads with PFS sequences. B) Distributions of RNA editing efficiencies for all 4-N PFS combinations at two different editing sites F) Quantification of the percent editing of REPAIRv1 at Cluc W85 across all possible 3 base motifs. Values represent mean +/− S.E.M. Non-targeting guide is the same as in Fig. 2C. C) Heatmap of 5’ and 3’ base preferences of RNA editing at Cluc W85 for all possible 3 base motifs.

Figure 4. Correction of disease-relevant mutations with REPAIRv1

A) Schematic of target and guide design for targeting AVPR2 878G>A. B) The 878G>A mutation (indicated by blue triangle) in AVPR2 is corrected to varying levels using REPAIRv1 with three different guide designs. For each guide, the region of duplex RNA is outlined in red. Values represent mean +/− S.E.M. Non-targeting guide is the same as in Fig. 2C. C) Schematic of target and guide design for targeting FANCC 1517G>A. D) The 1517G>A mutation (indicated by blue triangle) in FANCC is corrected to varying levels using REPAIRv1 with three different guide designs. For each guide, the region of duplex RNA is outlined in red. The heatmap scale bar is the same as in panel B. Values represent mean +/− S.E.M. Non-targeting guide is the same as in Fig. 2C. E) Quantification of the percent editing of 34 different disease-relevant G>A mutations selected from ClinVar using REPAIRv1. Non-targeting guide is the same as in Fig. 2C. F) Analysis of all the possible G>A mutations that could be corrected using REPAIR as annotated in the ClinVar database. G) The distribution of editing motifs for all G>A mutations in ClinVar is shown versus the editing efficiency by REPAIRv1 per motif as quantified on the Gluc transcript. Values represent mean +/− S.E.M.

Figure 5. Characterizing specificity of REPAIRv1

A) Schematic of KRAS target site and guide design. B) Quantification of percent A to I editing for tiled KRAS-targeting guides. Editing percentages are shown for the on-target (blue triangle) and neighboring adenosine sites. For each guide, the region of duplex RNA is outlined in red. Values represent mean +/− S.E.M. C) Transcriptome-wide sites of significant RNA editing by REPAIRv1 (150ng REPAIR vector transfected) with Cluc targeting guide. The on-target site Cluc site (254 A>I) is highlighted in orange. D) Transcriptome-wide sites of significant RNA editing by REPAIRv1 (150ng REPAIR vector transfected) with non-targeting guide. Non-targeting guide is the same as in Fig. 2C.

Figure 6. Rational mutagenesis of ADAR2 to improve the specificity of REPAIRv1

A) Quantification of luciferase signal restoration (on-target score, red boxes) by various dCas13-ADAR2_DD mutants as well as their specificity score (blue boxes) plotted along a schematic of the contacts between key ADAR2 deaminase residues and the dsRNA target (target strand shown in gray; the non-target strand is shown in red). All deaminase mutations were made on the dCas13-ADAR2_DD(E488Q) background. The specificity score is defined as the ratio of the luciferase signal between targeting guide and non-targeting guide conditions. Schematic of ADAR2 deaminase domain contacts with dsRNA is adapted from ref (20). B) Quantification of luciferase signal restoration by various dCas13-ADAR2 mutants versus their specificity score. Non-targeting guide is the same as in Fig. 2C. C) Quantification of on-target editing and the number of significant off-targets for each dCas13-ADAR2_DD(E488Q) mutant by transcriptome wide sequencing of mRNAs. Values represent mean +/− S.E.M. Non-targeting guide is the same as in Fig. 2C. D) Transcriptome-wide sites of significant RNA editing by REPAIRv1 (top) and REPAIRv2 (bottom) with a guide targeting a pretermination site in Cluc. The on-target Cluc site (254 A>I) is highlighted in orange. 10 ng of REPAIR vector was transfected for each condition. E) Representative RNA sequencing reads surrounding the on-target Cluc editing site (254 A>I; blue triangle) highlighting the differences in off-target editing between REPAIRv1 (top) and REPAIRv2 (bottom). A>I edits are highlighted in red; sequencing errors are highlighted in blue. Gaps reflect spaces between aligned reads. Non-targeting guide is the same as in Fig. 2C. F) RNA editing by REPAIRv1 and REPAIRv2 with guides targeting an out-of-frame UAG site in the endogenous KRAS and PPIB transcripts. The on-target editing fraction is shown as a sideways bar chart on the right for each condition row. For each guide, the region of duplex RNA is outlined in red. Values represent mean +/− S.E.M. Non-targeting guide is the same as in Fig. 2C.