Similar Datasets
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:Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level
| PRJNA769274 | ENA
Project description:Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level [transcriptome]
| PRJNA769277 | ENA
Project description:Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level [WGS]
| PRJNA769278 | ENA
Project description:A nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins, resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass, that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.
| S-EPMC6698196 | biostudies-literature
Project description:Adenine base editors (ABEs) have been exploited to introduce targeted adenine (A) to guanine (G) base conversions in various plant genomes, including rice, wheat and Arabidopsis. However, the ABEs reported thus far are all quite inefficient at many target sites in rice, which hampers their applications in plant genome engineering and crop breeding. Here, we show that unlike in the mammalian system, a simplified base editor ABE-P1S (Adenine Base Editor-Plant version 1 Simplified) containing the ecTadA*7.10-nSpCas9 (D10A) fusion has much higher editing efficiency in rice compared to the widely used ABE-P1 consisting of the ecTadA-ecTadA*7.10-nSpCas9 (D10A) fusion. We found that the protein expression level of ABE-P1S is higher than that of ABE-P1 in rice calli and protoplasts, which may explain the higher editing efficiency of ABE-P1S in different rice varieties. Moreover, we demonstrate that the ecTadA*7.10-nCas9 fusion can be used to improve the editing efficiency of other ABEs containing SaCas9 or the engineered SaKKH-Cas9 variant. These more efficient ABEs will help advance trait improvements in rice and other crops.
| S-EPMC7004905 | biostudies-literature
Project description:Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38-82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.
| S-EPMC8501978 | biostudies-literature
Project description:Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a β-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes.
| S-EPMC11022790 | biostudies-literature
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