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Cited by 573CRISPR and Genetic EngineeringRNA regulation and diseaseDNA Repair Mechanisms

CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells

Open access
Kurt, Ibrahim C., Zhou, Ronghao, Iyer, Sowmya, Garcia, Sara P., Miller, Bret R., Langner, Lukas M., Grünewald, Julian, Joung, J. Keith·2021·Nature Biotechnology
DOI 10.1038/s41587-020-0609-xPMC PMC7854778
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Abstract

CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications 1-4 but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities 5,6. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.

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Crossref · 10.1038/s41587-020-0609-x

Cites

9 of 9 references verified0 citation errors · 0 not checkable

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  • In current study, alternative base editing outcomes with SaKKH-CBE were occasionally observed, likely caused by accessibility issues of UGI to uracil in R-loop generated by SaKKH Cas9.

    “Of note, in the current study, we occasionally observe these alternative base editing outcomes with SaKKH-CBE, which is likely caused by accessibility issues of UGI to the uracil in the R-loop generated by SaKKH Cas964.”

    From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [64]

    The paper’s discussion of ABEmax editing (section labeled Abstract; page unavailable) reports unexpected C-to-G edits and shows that adding UGI domains reduced those edits, supporting a general link between UGI activity and alternative editing outcomes. It does not identify limited UGI access to uracil in a SaKKH Cas9-generated R-loop as the cause. The SaKKH-CBE observation is presented as the citing authors’ own result, not a result of this source. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Recent developments have resulted in C > G base editors that use cytidine deaminases with additional fusion proteins that drive repair of induced U towards G instead of T.

    “Indeed, recent developments have resulted in C > G base editors that use cytidine deaminases in combination with additional fusion proteins that drive repair of the induced U in the DNA towards G instead of T64.”

    From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [64]

    The supplied Abstract passages describe a C-to-G editor combining an APOBEC1 cytidine deaminase with a fused uracil DNA N-glycosylase, and report C-to-G editing. But they describe preferential introduction of G after uracil excision as a hypothesis involving an undefined mechanism; the paper says it lacks a mechanistic understanding of how C-to-G edits occur. The claim presents that repair pathway as established. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

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Cites

Flagged first, then by citations

Research works printed but not linked: 13

  1. [1]Rees HA & Liu DR Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet 19, 770– 788 (2018). 30323312 PMC6535181
  2. [6]Doman JL, Raguram A, Newby GA & Liu DR Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat Biotechnol (2020). PMC7335424 32042165
  3. [7]Grünewald J. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat Biotechnol 37, 1041– 1048 (2019). 31477922 PMC6730565
  4. [8]Kim HS, Jeong YK, Hur JK, Kim JS & Bae S. Adenine base editors catalyze cytosine conversions in human cells. Nat Biotechnol 37, 1145– 1148 (2019). 31548727
  5. [9]Komor AC Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv 3, eaao4774 (2017). PMC5576876 28875174
  6. [10]Koblan LW Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol 36, 843– 846 (2018). 29813047 PMC6126947
  7. [11]Gehrke JM An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat Biotechnol 36, 977– 982 (2018). 30059493 PMC6181770
  8. [14]Kleinstiver BP High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490– 495 (2016). 26735016 PMC4851738
  9. [15]Anzalone AV Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149– 157 (2019). 31634902 PMC6907074
  10. [17]Hu JH Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57– 63 (2018). 29512652 PMC5951633
  11. [19]Guo C. Transversions have larger regulatory effects than transitions. BMC Genomics 18, 394 (2017). 28525990 PMC5438547
  12. [20]Kim YB Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol 35, 371– 376 (2017). 28191901 PMC5388574
  13. [22]Clement K. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37, 224– 226 (2019). 30809026 PMC6533916

Cited by

Sorted by citations

Authors
Kurt, I. C., Zhou, R., Iyer, S., Garcia, S. P., Miller, B. R., Langner, L. M., Grünewald, J., & Joung, J. K.
Affiliations
  • Harvard University (US)
  • Massachusetts General Hospital (US)
Corresponding author
  • Grünewald, Julian
  • Joung, J. Keith
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2020-07-20
Volume
39
Issue
1
Pages
41-46
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/s41587-020-0609-x
PMC
PMC7854778
OpenAlex
W3043143974
Cited by
573
References
9
Citations by year
2026: 56 · 2025: 83 · 2024: 107 · 2023: 126 · 2022: 100 · 2021: 84 · 2020: 16 · 2012: 1
FWCI
26.77
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • FreiDok plus (Universitätsbibliothek Freiburg) (submittedVersion)
Funders
  • Boehringer Ingelheim Fonds MD fellowship
  • Deutsche Forschungsgemeinschaft (416375182)
  • U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (RM1 HG009490)
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (R35 GM118158)
  • United States Department of Defense | Defense Advanced Research Projects Agency (HR0011-17-2-0042)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Keywords
cytosine base editors, adenine base editors, uracil-DNA glycosylase, Cas9 nickase, AT-rich sequences, human cells, genome editing
MeSH terms
Gene Editing, Cytidine Deaminase, Cytosine, DNA, Guanine, Humans, HEK293 Cells, CRISPR-Cas Systems

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