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Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level [WGS]

ABSTRACT: 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.

ORGANISM(S): Oryza sativa

PROVIDER: GSE185495 | GEO | 2022/01/01

REPOSITORIES: GEO

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Comprehensive evaluation of adenine base editors induced mutations at genomic and transcriptional level [transcriptome]

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

Genome-wide specificity of genome editing by both CRISPR-Cas9 and TALEN in the model plant Physcomitrium patens

Project description:CRISPRs and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of SNVs and InDels for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and that the PEG treatment in itself was probably the main source of mutations found in the edited plants.

2022-03-03 | E-MTAB-11497 | biostudies-arrayexpress

Tumour mutations in long noncoding RNAs enhance cell fitness

Project description:Tumour DNA contains thousands of somatic single nucleotide variants (SNVs) in non-protein-coding elements, yet their functional significance remains poorly understood. Amongst the most highly mutated elements are long noncoding RNAs (lncRNAs), functional transcripts with known roles in carcinogenesis. To search for driver mutations in lncRNAs, we apply an integrative driver discovery algorithm to SNVs from 2583 primary tumours and 3527 metastases to reveal 54 potential “driver lncRNAs”. Our algorithm confirms a particularly high mutation rate in the iconic cancer lncRNA, NEAT1, which has been ascribed by recent studies to passenger effects. We directly test the functionality of NEAT1 SNVs using in cellulo mutagenesis, identifying discrete regions where mutations reproducibly increase cell proliferation in diverse cell backgrounds, both cancerous and normal. In particular, mutations in the 5’ region alter ribonucleoprotein assembly and boost the population of subnuclear paraspeckles, thus mechanistically linking mutations to cellular proliferation. We then used RNA-pull down followed by mass spectrometry to identify the protein interactor changing between the wild type and mutant form of NEAT1.

2023-07-20 | PXD034007 | Pride

Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq

Project description:Whole genome re-sequencing is still a costly method to detect genetic mutations that lead to altered forms of proteins and may be associated with disease development. Since the majority of disease-related single nucleotide variations (SNVs) are found in protein-coding regions, we propose to identify SNVs in expressed exons of the human genome using the recently developed RNA-Seq technique. We identify 12,176 and 10,621 SNVs, respectively, in Jurkat T cells and CD4+ T cells from a healthy donor. Interestingly, our data show that one copy of the TAL-1 protooncogene has a point mutation in 3’ UTR and only the mutant allele is expressed in Jurkat cells. We provide a comprehensive dataset for further understanding the cancer biology of Jurkat cells. Our results indicate that this is a cost-effective and efficient strategy to systematically identify SNVs in the expressed regions of the human genome. RNA-Seq experiments for two samples: CD4+ T cells from a healthy donor and Jurkat T cells. There are 8 lanes of data for each sample.

2009-06-15 | E-GEOD-16190 | biostudies-arrayexpress

The position and complex genomic architecture of plant T-DNA insertions revealed by 4C

Project description:The integration of T-DNA to plant genomes is widely used for basic research and agriculture. High heterogeneity in the number of integration events per genome, their configuration and impact on genome integrity highlight both, the critical need and great challenge to detect the genomic locations of T-DNA insertions and their associated chromosomal rearrangements. Here we present ‘4SEE’, a circular chromosome conformation capture (4C) based method for robust, rapid and cost-efficient detection of the entire scope T-DNA locations. Moreover, by measuring the chromosomal architecture at plant genome flanking the T-DNA insertions, 4SEE outlines their associated complex chromosomal aberrations. Applying 4SEE to a collection of confirmed T-DNA lines revealed previously unmapped T-DNA insertions and chromosomal rearrangements such as inversions and translocations. Uncovering such events in feasible, robust and cost-effective manners by 4SEE in any plant of interest have implications for accurate annotation and phenotypic characterization of T-DNA insertion mutants and transgene expression in basic science applications as well as for plant biotechnology.

2020-04-05 | GSE147542 | GEO

moue, rat tissue and human cell line

Project description:on and off target deep seq for ABEs

| PRJNA433929 | ENA

Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq

2009-06-15 | GSE16190 | GEO

Whole-genome sequencing of the CRISPR/Cas9-generated cell lines G12LS and C07 carrying a heterozygous and homozygous mutation in the RB1 gene and their parental human embryonic stem cell line H9.

Project description:Heterozygous and homozygous mutations were introduced to the human embryonic stem cell line H9 by using the CRISPR/Cas9-system. Since off-target effects can occur and high numbers of SNVs can be acquired during clonal selection, the generated cell lines and the parental cell line were analyzed by whole-genome sequencing.

2021-12-31 | E-MTAB-10320 | biostudies-arrayexpress

Cistrome-partitioning reveals convergence of somatic mutations and risk-variants on master transcription regulators in primary prostate tumors

Project description:Thousands of noncoding somatic Single Nucleotide Variants (SNVs) of unknown function are reported in tumors. Partitioning the genome according to cistromes, reveals the enrichment of somatic SNVs in prostate tumors as opposed to adjacent normal tissue cistromes of master transcription regulators, including AR, FOXA1 and HOXB13. This parallels enrichment of prostate cancer genetic predispositions over these transcription regulators’ tumor cistromes, exemplified at the 8q24 locus harboring both risk-variants and somatic SNVs in cis-regulatory elements, upregulating MYC expression and altering the binding of transcription regulators to DNA. However, Massively-Parallel Reporter Assays reveal that few SNVs can alter the transactivation potential of individual CREs. Instead, SNVs accumulate, similarly to inherited riskvariants, in cistromes of master transcription regulators required for prostate cancer development. Significance Difficulties in inferring the biological significance of noncoding mutations have limited their inclusion in precision genomics medicine pipelines. Most attempts to delineate a role for noncoding mutations relied on detecting evidence for positive selection within individual CREs, such as reported for the TERT gene promoter. By considering the enrichment of noncoding mutations in cistromes as opposed to individual CREs, we reveal their specificity towards master transcription regulators that promote prostate cancer development, a feature shared with inherited risk-variants. Overall, our work provides a blueprint for the functional interpretation of noncoding mutations in genomic tests relying on defining cis-regulatory units according to cistrome-partitioning to identify cancer driver transcription regulators.

2019-12-01 | GSE137527 | GEO

Directed Evolution of an Adenine Base Editor withIncreased Activity and Context Compatibility [sgRNA-target library]

Project description:Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. ABEs function most effectively when the target A is in a TA context. Deficient ABE processing of RA (R = A or G) is most evident when the target A is outside the comfortable editing window or when delivery is suboptimal. In the current study, we report directed evolution of TadA8r, a new variant of the Escherichia coli tRNA-specific adenosine deaminase (TadA) with ultra-fast deoxyadenosine deamination and no context bias.

2023-09-14 | GSE242012 | GEO

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