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Hypercompact adenine base editors based on a Cas12f variant guided by engineered RNA

Nature Chemical Biology volume 18, pages 1005–1013 (2022)Cite this article

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Matters Arising to this article was published on 16 February 2023

Matters Arising to this article was published on 16 February 2023

An Author Correction to this article was published on 16 February 2023

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Abstract

Cas12f is a hypercompact type V, Cas12 family member. Previously, we reported a set of engineered guide RNAs supporting high indel efficiency for Cas12f1 in human cells. Here we suggest a new technology whereby the engineered guide RNAs also manifest high-efficiency programmable endonuclease activity for a Cas12f variant. We have termed this technology TaRGET (Tiny nuclease RNA-based Genome Editing Technology). Having this feature in mind, we established TaRGET-based adenine base editors (ABEs). A Tad–Tad mutant (V106W, D108Q) dimer fused to the C terminus of dCas12f (D354A) showed the highest levels of A-to-G conversion. The limited targetable sites for TaRGET-ABE were expanded with engineered variants of Cas12f or optimized deaminases. Delivery of TaRGET-ABE also ensured potent A-to-G conversion rates in mammalian genomes. Collectively, the TaRGET-ABE will contribute to improving precise genome-editing tools that can be delivered by adeno-associated viruses, thereby harnessing the development of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene therapy.

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Fig. 1: Augment RNA-guided programmable nuclease using transposase B.
Fig. 2: Feasibility test and optimization of the TaRGET-ABE system.
Fig. 3: Expanding targetable sites by PAM variants of CWCas12f engineering.
Fig. 4: Switching a base-editing window by the engineering of Tad and CWCas12f.
Fig. 5: Validation of base-editing activity of the TaRGET-ABE-C3.0 system through AAV delivery in vitro.
Fig. 6: Assessment of base-editing activity in vivo via AAV delivery and off-target property of TaRGET-ABE system.

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Data availability

Data that support the findings of this study are available within the Article and its Supplementary Information. Deep-sequencing data for large-scale validation and RNA-seq data were deposited at the NCBI Sequence Read Archive database (http://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA823884. All other data that support the findings of the present study and plasmid vectors are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

Reditools is available at https://github.com/BioinfoUNIBA/REDItools2. MAUND is available at https://github.com/ibs-cge/maund.

Change history

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Acknowledgements

This work was supported by a grant through the KRIBB Research Initiative Program (KGM5382221 to Y.L., D.J., K.-H.P, H.J.C, J.M.L, J.-H.K and Y.-S.K.), the National Research Foundation of Korea (INNOPOLIS) grant (2021-DD-RD-0178-01 to D.Y.K., Y.C., S.P. and Y.-S.K.), and the National Research Foundation of Korea grant (2022R1C1C1013085 to D.Y.K and S.K.) funded by the Ministry of Science and ICT, the ‘Alchemist Project’ funded by the Ministry of Trade, Industry and Energy (20012445 to D.Y.K. and Y.-S.K.), and Cooperative Research Program for Agriculture Science and Technology Development (PJ0165422022 to D.Y.K., Y.C., S.P. and Y.-S.K.) funded by Rural Development Administration.

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Author notes

  1. These authors contributed equally: Do Yon Kim, Yuhee Chung.

Authors and Affiliations

  1. GenKOre, Daejeon, Republic of Korea

    Do Yon Kim, Yuhee Chung, Seyeon Park, Sumin Ko & Yong-Sam Kim

  2. KRIBB School of Bioscience, Korea University of Science and Technology, Daejeon, Republic of Korea

    Yujin Lee, Dongmin Jeong, Jeong-Heon Ko & Yong-Sam Kim

  3. Genome Editing Research Center, KRIBB, Daejeon, Republic of Korea

    Yujin Lee, Dongmin Jeong, Kwang-Hyun Park, Hyun Jung Chin, Jeong Mi Lee, Jeong-Heon Ko & Yong-Sam Kim

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Contributions

Y.-S.K. conceived the study and designed the experiments. D.Y.K. and Y.C. performed overall experiments. D.J and Y.L. constructed the PAM library vectors and PAM variants of CWCas12f. H.J.C. and J.M.L performed base-editing assays in cells. S.P. and S.K. derived PAM-mutant HEK293T cells. K.-H.P performed the structural analysis for CWCas12f engineering. J.-H.K. and Y.-S.K. interpreted data. Y.-S.K. wrote the manuscript.

Corresponding author

Correspondence to Yong-Sam Kim.

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Competing interests

D.Y.K., Y.C. and Y.-S.K. have filed patent applications on the TaRGET-ABE and PAM variants of CWCas12f through GenKOre. Y.-S.K. and D.Y.K. are co-founders of GenKOre. The remaining authors declare no other competing interests.

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Nature Chemical Biology thanks Sangsu Bae, Rahul Kohli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Construction of catalytically inactive CWCas12f (dCWCas12f) and confirmation of indel-null activity in HEK293T cells.

a. An agarose gel image showing cleavage pattern of plasmid vector by wild-type or several catalytically dead mutants of CWCas12f. Plasmid vectors were constructed to harbor protospacer and PAM sequence for sgRNA. Five microgram of plasmid vectors were incubated with 1 microgram of gRNA and 2 microgram of CWCas12f or dCWCas12f at 37 °C for 1 h. The cleaved vector samples were resolved on a 0.8% agarose gel. M denotes molecular ladders. The image corresponds to a representative experiment for three independent experiments. b. Indel efficiencies of CWCas12f or dead mutants of CWCas12f at an NLRC4 locus (5’-TTTAGAGGGAGACACAAGTTGATA-3’) in HEK293T cells. n = 3 independent experiments.

Extended Data Fig. 2 Dimerization of CWCas12f in the presence of single-stranded guide RNA.

a. An SDS-PAGE gel image for purified CWCas12f in elution fractions. The gel image corresponds to a representative experiment for six independent experiments. b. Size-exclusion chromatography profiles of CWCas12f proteins in the presence or absence of sgRNA. CWCas12f proteins were incubated with gRNA and the RNP complex was resolved on a Superdex 200 column. The molecular mass was estimated from a standard curve derived from the mobility of molecular ladder proteins.

Source data

Extended Data Fig. 3 Identification of the base-editing window of TaRGET-ABE-C2 system.

The substitution profile of TaRGET-ABE-C2 was explored for five endogenous sites in HEK293T cells to define a base editing window. The endogenous sites were selected from the validated sites showing substantial A-to-G conversion activities and also carrying multiple adenine sequences in the PAM-proximal region. The TaRGET-ABE system elicits A-to-G conversions in the range of positions 2–6, but most predominantly at the positions 3-4. The intensity of colors is not proportional to the editing efficiency, but highlights the qualitatively high efficiency positions.

Extended Data Fig. 4 Head-to-head comparison of TaRGET-based adenine base editors and Un1Cas12f1-based ABEMINI.

The efficiency of TaRGET-ABEs was compared with that of ABEMINI at five endogenous loci in HEK293T cells. Specifically, TaRGET-ABE-C2 was compared with ABEMINI to rule out the contribution of Tad engineering to the editing efficiency. The results show significant difference in A-to-G conversion activities in the base-editing window between TaRGET-ABE-C2 and ABEMINI, indicating CWCas12f as a preferred nuclease for hypercompact adenine base editors. The intensity of colors represents of the conversion efficiency. The values represent the means of three independent experiments.

Supplementary information

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Source Data Fig. 3

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Source Data Fig. 4

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Source Data Fig. 5

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Source Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 2

Unprocessed gel image.

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Kim, D.Y., Chung, Y., Lee, Y. et al. Hypercompact adenine base editors based on a Cas12f variant guided by engineered RNA. Nat Chem Biol 18, 1005–1013 (2022). https://doi.org/10.1038/s41589-022-01077-5

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