Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22860900_1.fastq.gz | Fastqsanger.gz | |||
| SRR22860900_2.fastq.gz | Fastqsanger.gz | |||
| SRR22861000_1.fastq.gz | Fastqsanger.gz | |||
| SRR22861000_2.fastq.gz | Fastqsanger.gz | |||
| SRR22861100_1.fastq.gz | Fastqsanger.gz |
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Project description:CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPC). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into CD3δ SCID patient’s HSPCs resulted in 71.2±7.85% (n=3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate preclinical efficacy of ABE in HSPC for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.
2023-03-20 | GSE220611 | 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:A-T to G-C base editing efficiency at targeted gene sites in HEK293T cells using the dCas12f-ABE design or the Cas12f-ABE design. Found that the total A-T to G-C conversion efficiency of Circular gRNAs exhibited about two-fold increase compared with U6 gRNAs. We further analyzed the pattern for A-T to G-C conversion on the target site, and observed that the most efficient base editing occurred in a narrow window A3 (3bp downstream of the PAM) similar to U6 gRNAs. In summary, Circular gRNAs with dCas12f-ABE design could enhance A-T to G-C base editing efficiency in a narrow window.
2024-03-11 | GSE260967 | GEO
Project description:Background The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigated the potential off-target structural variations associated with CRISPR/Cas9, ABE and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. Results The results showed that both Cas9 and ABE generated off-target structural variations (SVs) in mouse embryos, while CBE induced rare SVs. In addition, off-target large deletions were detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activated the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulated cell cycle-related genes and arrested in G0 phase. A percentage of 16.59% and 4.29% aneuploid cells were still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE were universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs were significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). Conclusions This study raises urgent need for minimizing the off-target SVs of CRISPR/Cas9 and ABE.
2024-11-01 | GSE280767 | 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:Plant biotechnology needs new methods that accelerate design-build-test-learn cycles to develop new gene editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle –from design to results– within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors and used it to develop an optimized LbCas12a-ABE system. Sequential improvement of five components –NLS, crRNA, LbCas12a nuclease, adenine deaminase and linker– led to a systematic, stepwise increase in ABE activity at extrachromosomal GFP reporter (from 0.5% to 40%) and endogenous target sites. We confirmed the activity of LbCas12a-ABE in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE. Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants.
2022-11-21 | GSE200450 | 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: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 most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by mutations in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia, and sudden cardiac death. The dominant-negative c.1208 G>A (p.R403Q) mutation in b-myosin (MYH7) is a common and well-studied mutation that leads to increased cardiac contractility and HCM onset. Here we identify an adenine base editor (ABE) and single-guide RNA system that can efficiently correct this human pathogenic mutation with minimal off-target and bystander editing. We show that delivery of base editing components rescues pathological manifestations of HCM in iPSC-cardiomyocytes derived from HCM patients and in a humanized mouse model of HCM. Our findings demonstrate the use of base editing to treat inherited cardiac diseases and prompt the further development of ABE-based therapies to correct a variety of monogenic mutations causing cardiac disease.
2022-10-25 | GSE201755 | GEO