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A cytosine deaminase for programmable single-base RNA editing

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Science  26 Jul 2019:
Vol. 365, Issue 6451, pp. 382-386
DOI: 10.1126/science.aax7063
  • Fig. 1 Evolution of an ADAR2 deaminase domain for cytidine deamination.

    (A) Schematic of RNA targeting of the catalytic residue mutant (C82R) of Gaussia luciferase reporter transcript. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (B) Heatmap depicting the percent editing levels of RESCUEr0 to RESCUEr16 on cytidines flanked by varying bases on the Gluc transcript. More favorable editing motifs are shown in the top map, whereas less favorable motifs (5′ C) are shown at the bottom. (C) Editing activity of RESCUE on all possible 16 cytidine flanking-bases motifs on the Gluc transcript with U-flip or C-flip guides. (D) Activity comparison between RESCUE, ADAR2dd without Cas13, full-length ADAR2 without Cas13, or no protein. (E) Editing efficiency of RESCUE on a panel of endogenous genes covering multiple motifs. The best guide for each site is shown, with the entire panel of guides displayed in fig. S19. Error bars in (C) to (E) indicate ±SEM (n = 3 replicates).

  • Fig. 2 Phenotypic outcomes of RESCUE on cell growth and signaling.

    (A) Schematic of β-catenin domains and RESCUE targeting guide. (B) Schematic of β-catenin activation and cell growth via RESCUE editing. (C) Percent editing by RESCUE at relevant positions in the CTNNB1 transcript. (D) Activation of Wnt/β-catenin signaling by RNA editing as measured by β-catenin–driven (TCF/LEF) luciferase expression. (E) Representative microscopy images of RESCUE CTNNB1 targeting and nontargeting guides in HEK293FT cells. (F) Quantitation of cellular growth due to activation of CTNNB1 signaling by RNA editing in HEK293FT cells. Error bars in (C), (D), and (F) indicate ±SEM (n = 3). *P < 0.05; ns, not significant; NT, nontargeting guide.

  • Fig. 3 RESCUE and REPAIR multiplexing and specificity enhancement via guide engineering.

    (A) Schematic of multiplexed C-to-U and A-to-I editing with pre-crRNA guide arrays. (B) Simultaneous C-to-U and A-to-I editing on CTNNB1 transcripts. (C) Schematic of rational engineering with guanine base-flips to prevent off-target activity at neighboring adenosine sites. (D) Percent editing at on-target C and off-target A sites for Gaussia luciferase (left) and KRAS (right) using rational introduction of disfavored base-flips. Error bars in (B) and (D) indicate ±SEM (n = 3).

  • Fig. 4 Transcriptome-wide specificity of RESCUE.

    (A) On-target C-to-U editing and summary of C-to-U and A-to-I transcriptome-wide off-targets for RESCUE compared to REPAIR. (B) Manhattan plots of RESCUE A-to-I (left) and C-to-U (right) off-targets. The on-target C-to-U edit is depicted in orange. (C) Schematic of ADAR2dd interactions with RNA. Residues mutated for improving specificity are depicted in red. (D) Luciferase values for C-to-U activity with a targeting guide (y axis) and A-to-I activity with a nontargeting guide (x axis) shown for RESCUE and 95 RESCUE mutants. RESCUE is depicted in red and mutants with better specificity in blue. The T375G mutant (REPAIRv2) is shown in orange. (E) On-target C-to-U editing and summary of C-to-U and A-to-I transcriptome-wide off targets of RESCUE, REPAIR, and highest-specificity mutants. (F) Manhattan plots of RESCUE-S (+S375A) A-to-I (left) and C-to-U (right) off-targets. The on-target C-to-U edit is shown in orange. (G) Representative RNA-sequencing reads surrounding the on-target Gluc editing site (blue triangle) for RESCUE (left) and RESCUE-S (right). A-to-I edits are depicted in red; C-to-U (T) edits are shown in blue; sequencing errors are depicted in yellow. Error bars in (D) and (E) indicate ±SEM (n = 3).

Supplementary Materials

  • A cytosine deaminase for programmable single-base RNA editing

    Omar O. Abudayyeh, Jonathan S. Gootenberg, Brian Franklin, Jeremy Koob, Max J. Kellner, Alim Ladha, Julia Joung, Paul Kirchgatterer, David B. T. Cox, Feng Zhang

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S35
    • Tables S1 to S8
    • References
    Structure S1

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