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A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing.

ABSTRACT: Existing adenine and cytosine base editors induce only a single type of modification, limiting the range of DNA alterations that can be created. Here we describe a CRISPR-Cas9-based synchronous programmable adenine and cytosine editor (SPACE) that can concurrently introduce A-to-G and C-to-T substitutions with minimal RNA off-target edits. SPACE expands the range of possible DNA sequence alterations, broadening the research applications of CRISPR base editors.

SUBMITTER: Grunewald J

PROVIDER: S-EPMC7723518 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing.

Grünewald Julian J Zhou Ronghao R Lareau Caleb A CA Garcia Sara P SP Iyer Sowmya S Miller Bret R BR Langner Lukas M LM Hsu Jonathan Y JY Aryee Martin J MJ Joung J Keith JK

Nature biotechnology 20200601 7

Existing adenine and cytosine base editors induce only a single type of modification, limiting the range of DNA alterations that can be created. Here we describe a CRISPR-Cas9-based synchronous programmable adenine and cytosine editor (SPACE) that can concurrently introduce A-to-G and C-to-T substitutions with minimal RNA off-target edits. SPACE expands the range of possible DNA sequence alterations, broadening the research applications of CRISPR base editors. ...[more]

PMID: 32483364

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Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos.

Project description:Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.

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A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.

Project description:Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.

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CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1.

Project description:BackgroundCRISPR/Cas9 systems have been repurposed as canonical genome editing tools in a variety of species, but no application for the model strain Rhodobacter sphaeroides 2.4.1 was unveiled.ResultsHere we showed two kinds of programmable base editing systems, cytosine base editors (CBEs) and adenine base editors (ABEs), generated by fusing endonuclease Cas9 variant to cytosine deaminase PmCDA1 or heterodimer adenine deaminase TadA-TadA*, respectively. Using CBEs, we were able to obtain C-to-T mutation of single and double targets following the first induction step, with the efficiency of up to 97% and 43%; while the second induction step was needed in the case of triple target, with the screening rate of 47%. Using ABEs, we were only able to gain A-to-G mutation of single target after the second induction step, with the screening rate of 30%. Additionally, we performed a knockout analysis to identify the genes responsible for coenzyme Q10 biosynthesis and found that ubiF, ubiA, ubiG, and ubiX to be the most crucial ones.ConclusionsTogether, CBEs and ABEs serve as alternative methods for genetic manipulation in Rhodobacter sphaeroides and will shed light on the fundamental research of other bacteria that are hard to be directly edited by Cas9-sgRNA.

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Project description:Adenine and cytosine base editors (ABEs and CBEs) represent a new genome editing technology that allows the programmable installation of A-to-G or C-to-T alterations on DNA. We engineered Streptococcus pyogenes Cas9-based adenine and cytosine base editor (SpACE) that enables efficient simultaneous introduction of A-to-G and C-to-T substitutions in the same base editing window on DNA.

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DNA capture by a CRISPR-Cas9-guided adenine base editor.

Project description:CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.

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A cytosine deaminase for programmable single-base RNA editing.

Project description:Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive β-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.

| S-EPMC6956565 | biostudies-literature

Small-molecule compounds boost genome-editing efficiency of cytosine base editor.

Project description:Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.

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Cytosine Base Editor (hA3A-BE3-NG)-Mediated Multiple Gene Editing for Pyramid Breeding in Pigs.

Project description:Pig is an important agricultural economic animal, providing large amount of meat products. With the development of functional genomics and bioinformatics, lots of genes and functional single nucleotide polymorphisms (SNPs) related to disease resistance and (or) economic traits in pigs have been identified, which provides the targets for genetic improvement by genome editing. Base editors (BEs), combining Cas9 nickase and cytidine or adenine deaminase, achieve all four possible transition mutations (C-to-T, A-to-G, T-to-C, and G-to-A) efficiently and accurately without double strand breaks (DSBs) under the protospacer adjacent motif (PAM) sequence of NGG. However, the NGG PAM in canonical CRISPR-Cas9 can only cover approximately 8.27% in the whole genome which limits its broad application. In the current study, hA3A-BE3-NG system was constructed with the fusion of SpCas9-NG variant and hA3A-BE3 to create C-to-T conversion at NGN PAM sites efficiently. The editing efficiency and scope of hA3A-BE3-NG were confirmed in HEK293T cells and porcine fetal fibroblast (PFF) cells. Results showed that the efficiency of hA3A-BE3-NG was much higher than that of hA3A-BE3 on NGH (H = A, C, or T) PAM sites (21.27 vs. 2.81% at average). Further, nonsense and missense mutations were introduced efficiently and precisely via hA3A-BE3-NG in multiple pig economic trait-related genes (CD163, APN, MSTN, and MC4R) in PFF cells by one transfection. The current work indicates the potential applications of hA3A-BE3-NG for pyramid breeding studies in livestock.

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Bimodal Adenine and Cytosine CRISPR Base Editing on DNA

Project description:Bimodal Adenine and Cytosine CRISPR Base Editing on DNA

| PRJNA565401 | ENA

Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing.

Project description:Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks. However, both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Therefore, the potential effects of deaminases induced by DNA base editors are of great importance for their clinical applicability. Here, the transcriptome-wide deaminase effects on gene expression and splicing is examined. Differentially expressed genes (DEGs) and differential alternative splicing (DAS) events, induced by base editors, are identified. Both CBEs and ABEs generated thousands of DEGs and hundreds of DAS events. For engineered CBEs or ABEs, base editor-induced variants had little effect on the elimination of DEGs and DAS events. Interestingly, more DEGs and DAS events are observed as a result of over expressions of cytosine and adenine deaminases. This study reveals a previously overlooked aspect of deaminase effects in transcriptome-wide gene expression and splicing, and underscores the need to fully characterize such effects of deaminase enzymes in base editor platforms.

| S-EPMC8285404 | biostudies-literature

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