Repair of CRISPR-guided RNA breaks enables site-specific RNA editing in human cells

Abstract

Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. Here, we show that site-specific RNA breaks generated with RNA-targeting CRISPR complexes are repaired in human cells, and this repair can be used for programmable deletions in human transcripts that restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.

Fig. 1.. Programmable deletions of RNA with RNA-guided type III-A CRISPR complexes.

A) Diagram of RNA editing in eukaryotic cells through sequence-specific RNA cleavage and RNA repair. B) Human cells (293T) were transfected with plasmids encoding for type III CRISPR complex of Streptococcus thermophilus (SthCsm), and RNA guides targeting PPIB or PARK7 messenger RNAs. Target transcripts were quantified with RT-qPCR, and the qPCR signal was normalized to ACTB and non-targeting guide RNA control using the ΔΔCt method. Data is shown as mean ± one standard deviation of three biological replicates. C) Zoom-in from panel B. Deep sequencing was used to quantify the proportion of signal that is derived from edited RNA. D) Top: schematics of deep sequencing approach used to quantify RNA editing. Sequencing reads were aligned to the reference sequence, and modifications at the target site were quantified. Bottom: top five most frequent RNA editing outcomes in PARK7 transcript (guide 2). Dotted lines indicate the positions of RNA breaks by the SthCsm complex. Red dashes depict deletions (Δ) identified in the sequencing data. The proportion of reads with deletions is shown as mean ± one standard deviation of three biological replicates.

Fig. 2.. Depletion of human RNA ligase RTCB restricts the repair of type III CRISPR-mediated RNA breaks.

A) Human RNA ligase RTCB joins RNA ends with a 2′,3′-cyclic phosphate (2’,3’>P) and a 5′-hydroxyl (5’-OH) that are produced by cellular nucleases in tRNA splicing (top) and non-canonical XBP1 mRNA splicing (bottom) during the unfolded protein response. RNA cleavage by SthCsm generates a 2’,3’>P and a 5’-OH at each cut site, and we hypothesized that the ligase activity of RTCB is involved in the RNA repair identified in Fig. 1 (middle). B) Western blot with anti-RTCB or anti-ACTB (loading control) antibodies was performed with lysates of 293T cells with (+) or without (−) RTCB depletion. See the uncropped images in fig. S2. C) PARK7 transcript was targeted with SthCsm (guide 2) in 293T cells with (+) or without (−RTCB depletion. PARK7 transcript was quantified with RT-qPCR and normalized to ACTB non-targeting guide RNA control using the ΔΔCt method. Data are shown as the mean ± standard deviation of three biological replicates. Unequal variances t-test was used to compare mean values. *p-value < 0.05. D) qPCR products in (C) were sequenced, and resulting reads were aligned to the reference sequence of the PARK7 transcript (NM_007262, GenBank). Graphs show sequencing depth (y-axes) at the amplified region of the transcript (x-axes). Every line shows a biological replicate (n = 3). The horizontal black bar indicates a region complementary to the guide RNA of the SthCsm complex. Vertical dotted lines mark predicted positions of RNA breaks. E) Quantification of deletions in target region of the PARK7 transcript. Data is shown as the mean ± standard deviation of three biological replicates. Unequal variances t-test was used to compare mean values. **** p < 0.001. F) The distribution of different deletion outcomes in (D).

Fig. 3.. Programmable deletion of stop codons restores protein expression.

A) Schematic representation of the proposed approach for deleting premature stop codons in human transcripts. B) Top: schematic diagram of the stop-GFP reporter plasmid. Bottom: Six guide RNAs for SthCsm complex were designed to excise the stop codon in the gfp transcript. Underlined (red) sections of target RNA are expected to be deleted. Vertical red ticks indicate predicted sites for RNA breaks. C) Cells were transfected with plasmids for the stop-GFP reporter and SthCsm with a non-targeting guide (left), stop-GFP reporter and SthCsm with a targeting guide (middle), or GFP reporter and SthCsm with the non-targeting guide (right). Fluorescence microscopy was used to image cells 48 h post-transfection. D) Top: schematic diagram of the GFP-stop-Luc reporter plasmid. Bottom: Six crRNAs for SthCsm complex were designed to delete the stop codon at the 3’-end of the gfp gene. E) Luciferase activity was measured in cell lysates 48 hours after transfection with GFP-stop-Luc and SthCsm plasmids. Luciferase activity is normalized to a control transfected with a reporter plasmid without the stop codon. Data are shown as mean ± one standard deviation of three replicates. Means were compared using one-way ANOVA, and samples with targeting guide RNAs were compared to the non-targeting control using one-tailed Dunnett’s test. * p < 0.5, ** p < 0.1, *** p < 0.001. F) Most frequent RNA editing outcomes in the sample with the most efficient rescue of luciferase activity in (B) (guide 1). Editing efficiency was quantified as mean ± one standard deviation of three biological replicates. Black box shows the stop codon that was targeted by type III CRISPR complexes. G, H, I). The same as (D-F), but with DisCas7-11.