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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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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.

| S-EPMC7183636 | biostudies-literature

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.

| S-EPMC7381381 | biostudies-literature

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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Bimodal Adenine and Cytosine CRISPR Base Editing on DNA

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.

2020-07-02 | GSE137411 | GEO

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Project description:Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

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Adenine base editor incorporating the N-methylpurine DNA glycosylase MPGv3 enables efficient A-to-K base editing in rice.

Project description: Not available

| S-EPMC10721470 | biostudies-literature

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

Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli.

Project description:Genome-editing (GE) techniques like base editing are ideal for introducing novel gain-of-function mutations and in situ protein evolution. Features of base editors (BEs) such as higher efficacy, relaxed protospacer adjacent motif (PAM), and a broader editing window enables diversification of user-defined targeted locus. Cytosine (CBE) or adenine (ABE) BEs alone can only alter C-to-T or A-to-G in target sites. In contrast, dual BEs (ACBEs) can concurrently generate C-to-T and A-to-G modifications. Although BE tools have recently been applied in microbes, there is no report of ACBE for microbial GE. In this study, we engineered four improved ACBEs (iACBEs) tethering highly active CBE and ABE variants that can introduce synchronized C-to-T and A-to-G mutations in targeted loci. iACBE4 generated by evoCDA1-ABE9e fusion demonstrated a broader editing window (positions -6 to 15) and is also compatible with the multiplex editing approach in Escherichia coli. We further show that the iACBE4-NG containing PAM-relaxed nCas9-NG expands the targeting scope beyond NGG (N-A/G/C/T) PAM. As a proof-of-concept, iACBE was effectively utilized to identify previously unknown mutations in the rpoB gene, conferring gain-of-function, i.e., rifampicin resistance. The iACBE tool would expand the CRISPR-GE toolkit for microbial genome engineering and synthetic biology. IMPORTANCE Dual base editors are DSB-free CRISPR tools applied in eukaryotes but not yet in bacteria. We developed an improved ACBE toolset for bacteria, combining highly processive deaminases. We believe that the bacterial optimized iACBE toolset is a significant advancement in CRISPR-based E. coli genome editing and adaptable to other microbes.

| S-EPMC9973308 | biostudies-literature

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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Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.

Project description:Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.

| S-EPMC9630288 | biostudies-literature

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