Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1052081 | ENA |
REPOSITORIES: ENA
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Project description:CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
2019-07-11 | GSE129893 | GEO
Project description:CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
2019-07-11 | GSE129889 | GEO
Project description:Base editors (BEs) shed new light on correcting disease-related T-to-C mutations. However, current rat APOBEC1-based BEs are less efficient in editing cytosines in highly-methylated regions or in GpC context. By screening a variety of APOBEC/AID deaminases, we showed that human APOBEC3A-conjugated BE and its engineered forms can mediate efficient C-to-T base editing in all examined contexts, including regions with high-methylation levels and GpC dinucleotides, which extends base editing scope.
2018-08-20 | GSE114999 | GEO
Project description:Conjugation of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications, but suffers from off-target (OT) mutations. By taking advantage of a cleavable deoxycytidine deaminase inhibitor (dCDI) domain, a transformer BE (tBE) system is developed to induce efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, tBE remains inactive at OT sites with the fusion of a cleavable dCDI, thus eliminating unintended mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise base changes. After delivery into mice via a dual-AAV system, tBE created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9 level, which resulted in ~30-40% decrease of total cholesterol. Together, the development of tBE establishes a highly-precise base editing system and its in vivo efficacy envisions potential therapeutic applications.
2021-03-12 | GSE164837 | GEO
Project description:Conjugation of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications, but suffers from off-target (OT) mutations. By taking advantage of a cleavable deoxycytidine deaminase inhibitor (dCDI) domain, a transformer BE (tBE) system is developed to induce efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, tBE remains inactive at OT sites with the fusion of a cleavable dCDI, thus eliminating unintended mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise base changes. After delivery into mice via a dual-AAV system, tBE created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9 level, which resulted in ~30-40% decrease of total cholesterol. Together, the development of tBE establishes a highly-precise base editing system and its in vivo efficacy envisions potential therapeutic applications.
2021-03-12 | GSE164477 | GEO
Project description:Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.
2024-01-12 | GSE253182 | GEO
Project description:Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.
2024-01-12 | GSE253184 | GEO
Project description:Current base editors use DNA deaminases, including cytidine deaminase in cytidine base editor (CBE) or adenine deaminase in adenine base editor (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for base editors capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel base editors, but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.
2024-01-23 | GSE253655 | GEO
Project description:Emerging base and prime editing may provide safer and more precise genetic engineering than nuclease-based approaches bypassing the dependence on DNA double strand breaks (DSBs). However, little is known about cellular responses and genotoxicity. Here, we comparatively assessed state-of-the-art base and prime editors (B/PE) versus Cas9 in human hematopoietic stem/progenitor cells (HSPCs). BE and PE induced detrimental transcriptional responses constraining editing efficiency and/or HSPC repopulation in xenotransplants, albeit to a lesser extent than Cas9. DNA DSBs and their genotoxic byproducts, including deletions and translocations, were less frequent but not abrogated by BE and PE, particularly for cytidine BE due to suboptimal inhibition of base excision repair. Tailoring timing and B/PE expression enabled highly efficient and precise editing of long-term repopulating HSPCs. However, we uncovered a genome-wide effect of BEs on the mutational landscape of HSPCs, raising concerns for a potential genotoxic impact and calling for further investigations and improvements in view of clinical application.and/or HSPC repopulation in xenotransplants, albeit to a lesser extent than Cas9. DNA DSBs and their genotoxic byproducts, including deletions and translocations, were lower but not abrogated by BE and PE, particularly for cytidine BE upon suboptimal base excision repair inhibition. Tailoring timing and B/PE expression enabled highly efficient and precise editing of long-term repopulating HSPCs. However, we uncovered a genome-wide effect of cytidine BE on mutational landscape of hematopoietic grafts, raising concerns for its prospective clinical application. Conversely, the superior efficiency and precision of adenine BE built confidence on its entry into clinical arena.
2023-07-18 | GSE218462 | GEO