Directed Evolution of an Adenine Base Editor withIncreased Activity and Context Compatibility [sgRNA-target library]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE242012 | GEO | 2023/09/14
REPOSITORIES: GEO
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Project description:It is known that current the-art-of-the-state TadA8 and TadA8e which evolved from E. coli TadA. They inherited the 'YA' context from tRNA deaminase. We started with wildtype E. coli TadA and designed an evolution campaign to force TadA variants to deaminate “GA” with fast kinetics. Three rounds of de novo directed evolution followed by DNA shuffling led to TadA8r, a TadA variant of superior “RA” deamination activity. TadA8r acts on a broadened editing window when fused to Streptococcus pyogenes Cas9 (SpCas9) and delivers robust editing at PAM distal positions. While highly active at on-target sites, ABE8r shows off-target DNA and RNA editing much lower than ABE8e. The off-target effects of ABE8r can be further mitigated by introducing a V106W substitution23, a R153 deletion22, or by mRNA delivery. Lastly, we demonstrate ABE8r-mediated correction of G1961E in ABCA4, the most prevalent mutation driving Stargardt disease (STGD1), in a “GA” context. ABE8r, with its superior activity and broadened context compatibility, complements and expands the current ABE family.
2023-09-14 | GSE220469 | GEO
Project description:Adenine base editors (ABEs) with wide CRISPR compatibility and high activity improves the editing efficiency and arouses the off-target challenges as well. Here, we carried out a comprehensive evaluation of ABE8e and ABE9 induced DNA and RNA mutations in model organism rice. The whole-genome sequencing analysis on plants with rBE46b (SpCas9n-TadA8e), rBE49b (SpCas9n-TadA9), rBE50 (SpCas9n-NG-TadA8e), rBE53 (SpCas9n-NG-TadA9) reveals that the ABEs with TadA9 lead to a higher number of off-target A>G SNVs and ABEs with SpCas9n-NG lead to a higher total number of off-target SNVs. The analysis of T-DNAs (ABEs carrier) disclosed that the on-target mutations could happen before T-DNA integration to plant genomes as well as after T-DNA integration to plant genomes, while ABEs integrated into plant genomes lead to more A>G SNVs. We also detected off-target A>G RNA mutations in plants with higher expression of ABEs but not in plants with lower expression of ABEs. The off-target A>G RNA mutations tend to cluster while off-target A>G DNA mutations cluster in a very rare manner. The findings that CRISPRs, TadA variants, T-DNA integration, and ABE expression contribute ABEs’ specificity provide alternative ways to increase the specificity of ABEs.
2022-01-01 | GSE185496 | GEO
Project description:Adenine base editors (ABEs) with wide CRISPR compatibility and high activity improves the editing efficiency and arouses the off-target challenges as well. Here, we carried out a comprehensive evaluation of ABE8e and ABE9 induced DNA and RNA mutations in model organism rice. The whole-genome sequencing analysis on plants with rBE46b (SpCas9n-TadA8e), rBE49b (SpCas9n-TadA9), rBE50 (SpCas9n-NG-TadA8e), rBE53 (SpCas9n-NG-TadA9) reveals that the ABEs with TadA9 lead to a higher number of off-target A>G SNVs and ABEs with SpCas9n-NG lead to a higher total number of off-target SNVs. The analysis of T-DNAs (ABEs carrier) disclosed that the on-target mutations could happen before T-DNA integration to plant genomes as well as after T-DNA integration to plant genomes, while ABEs integrated into plant genomes lead to more A>G SNVs. We also detected off-target A>G RNA mutations in plants with higher expression of ABEs but not in plants with lower expression of ABEs. The off-target A>G RNA mutations tend to cluster while off-target A>G DNA mutations cluster in a very rare manner. The findings that CRISPRs, TadA variants, T-DNA integration, and ABE expression contribute ABEs’ specificity provide alternative ways to increase the specificity of ABEs.
2022-01-01 | GSE185495 | GEO
Project description:ABEs were developed to catalyze an A-to-G conversion, thus holding therapeutic potentials for treating the major class of human pathogenic SNPs. However, robust and precise editing at diverse genome loci in a controllable manner remains challenging. Here, through a high-throughput chemical screen of ~ 8,000 small molecules, we identified and validated a spectrum of small molecules that target the canonical TGF-beta pathway as ABE activators. Among those, SB505124, a selective ALK5 inhibitor, promotes ABE editing most. Treating cells with SB505124 dramatically enhanced on-target editing at multiple genome loci, including the refractory regions, while exhibiting little effect on off-target conversion on the genome, eliminating the major concern for clinical applications. Furthermore, SB505124 facilitates the editing of disease-associated genes in vitro and in vivo. Intriguingly, SB505124 serves as a specific activator by selectively promoting the activity of ABEs, rather than CBEs or Cas9. Our finding equips the ABE with precise chemical control, and more importantly, reveals a so-far-unreported role of the canonical TGF-beta pathway on gene editing.
2022-09-20 | GSE208182 | GEO
Project description:Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C>T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.
2022-08-25 | GSE211876 | GEO
Project description:The majority of known pathogenic point mutations in the human genome are C•G to T•A substitutions. Adenine base editors (ABEs), comprised of nuclease-impaired Cas9 fused to adenine deaminases, enable direct repair of these mutations, making them promising tools for precision in vivo genome editing therapies. However, prior to application in patients, thorough safety and efficacy studies in relevant model organisms are needed. Here, we apply adenine base editing in vivo in the liver of mice and cynomolgus macaques to install a splice site mutation in PCSK9 and reduce blood low-density lipoprotein (LDL) levels, a well-known risk factor for cardiovascular disease. Intravenous delivery of ABE-encoding mRNA and a locus-specific single guide (sg)RNA utilizing lipid nanoparticle (LNP) technology induce up to 67% editing in the liver of mice and up to 34% editing in the liver of macaques, leading to a reduction of plasma PCSK9 and LDL levels. We observed rapid clearance of ABE mRNA after LNP-mediated delivery, and neither sgRNA-dependent nor sgRNA-independent off-target mutations are detected in genomic DNA. Together, our findings support safety and feasibility of adenine base editing to treat patients with monogenetic liver diseases.
2021-04-13 | GSE168364 | GEO
Project description:The majority of known pathogenic point mutations in the human genome are C•G to T•A substitutions. Adenine base editors (ABEs), comprised of nuclease-impaired Cas9 fused to adenine deaminases, enable direct repair of these mutations, making them promising tools for precision in vivo genome editing therapies. However, prior to application in patients, thorough safety and efficacy studies in relevant model organisms are needed. Here, we apply adenine base editing in vivo in the liver of mice and cynomolgus macaques to install a splice site mutation in PCSK9 and reduce blood low-density lipoprotein (LDL) levels, a well-known risk factor for cardiovascular disease. Intravenous delivery of ABE-encoding mRNA and a locus-specific single guide (sg)RNA utilizing lipid nanoparticle (LNP) technology induce up to 67% editing in the liver of mice and up to 34% editing in the liver of macaques, leading to a reduction of plasma PCSK9 and LDL levels. We observed rapid clearance of ABE mRNA after LNP-mediated delivery, and neither sgRNA-dependent nor sgRNA-independent off-target mutations are detected in genomic DNA. Together, our findings support safety and feasibility of adenine base editing to treat patients with monogenetic liver diseases.
2021-04-13 | GSE168363 | GEO
Project description:To alleviate the ABE-mediated cytosine editing activity, we engineered the commonly-used version of adenosine deaminase, TadA7.10. We found that the D108Q mutation also reduces cytosine deamination activity in two recently-developed versions of ABE, ABE8e and ABE8s, and has a synergistic effect with V106W, a key mutation that reduces off-target RNA editing.
2021-01-21 | GSE165164 | 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