Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE98685 | GEO | 2017/08/29
SECONDARY ACCESSION(S): PRJNA385910
REPOSITORIES: GEO
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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:The targeting range of CRISPR-Cas9 base editors (BEs) is limited by their G/C-rich PAM sequences. To overcome this limitation, we developed a CRISPR/Cpf1-based BE by fusing the rat cytosine deaminase APOBEC1 to a catalytically inactive version of Lachnospiraceae bacterium Cpf1. The base editor recognizes a T-rich PAM sequence and converts C to T in human cells with low levels of indels, non-C-to-T substitutions and off-target editing.
2018-03-18 | GSE110136 | GEO
Project description:C-to-T base editing mediated by CRISPR/Cas9 base editors (BEs) needs a G/C-rich PAM and the editing fidelity is compromised by unwanted indels and non-C-to-T substitutions. We developed CRISPR/Cpf1-based BEs to recognize a T-rich PAM and induce efficient C-to-T editing with few indels and/or non-C-to-T substitutions. The requirement of editing fidelity in therapeutic-related trials necessitates the development of CRISPR/Cpf1-based BEs, which also facilitates base editing in A/T-rich regions.
2018-10-16 | GSE105002 | GEO
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
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:We show that delivering the mitochondrial base editor DdCBEs via AAV transduction of somatic cells efficiently produces precise base editing of the intended region.
2022-01-19 | GSE184064 | GEO
Project description:Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived human pluripotent stem cells (hPSCs). Unlike Cas9-mediated knock-in, cytosine base editor (CBE) and prime editor (PE) achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by CBE or PE additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of dominant negative p53 (p53DD) and three UNG inhibitor (UGI), engineered to specifically diminish base excision repair (BER), improved CBE efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional PE system also significantly enhanced PE efficiency in hPSCs. Thus, combined inhibition of the unique cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.
2024-04-22 | GSE247589 | GEO
Project description:Uracil DNA glycosylases (UDGs) excise uracil from DNA arising from dUMP misincorporation during replication or from cytosine deamination. Besides functioning in canonical uracil repair, UDGs cooperate with DNA base modifying enzymes to effect mutagenesis or DNA demethylation. Mammalian cells express four UDGs, the functional dissection of which represents a challenge. Here, we used Schizosaccharomyces pombe with only two UDGs, Ung1 and Thp1, as a simpler model to study functional interactions in uracil repair. We show that despite a predominance of Ung1 activity in cell extracts, both UDGs act redundantly against genomic uracil accumulation and mutations from cytosine deamination in cells. Notably, Thp1 but not Ung1-dependent repair is cytotoxic under genomic uracil stress induced by 5-fluorouracil exposure or AID expression. Also, Thp1- but not Ung1-mediated base excision is recombinogenic, accounting for more than 60% of spontaneous mitotic recombination events in a recombination assay. Hence, the qualitative outcome of uracil repair depends on the initiating UDG; while Ung1 shows expected features of a bona-fide DNA repair enzyme, Thp1-initiated repair appears slow and non-productive, implicating a function beyond canonical DNA repair. Given the epigenetic role of mammalian TDGs, we performed transcriptome analyses and identified a possible function of Thp1 in stabilizing gene expression.
2014-12-31 | GSE64585 | GEO
Project description:Highly efficient base editing in human zygotes based on SaKKH-BE3
| PRJNA439196 | ENA