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Accessing and Identification of Novel Environmental Alleles of the ACC Deaminase Domain Region through a Competition Assay

ABSTRACT: Accessing and Identification of Novel Environmental Alleles of the ACC Deaminase Domain Region through a Competition Assay

PROVIDER: PRJEB11480 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR1104482_1.fastq.gz	Fastqsanger.gz
	ERR1104482_2.fastq.gz	Fastqsanger.gz

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Transcription profiling of canola seedling roots and shoots to study the effects of the bacterial enzyme ACC deaminase

Project description:The purpose of this experiment was to study the effects of the bacterial enzyme ACC deaminase on the transcriptional changes within canola seedlings. Seedlings from seeds treated with the plant growth-promoting bacteria Pseudomonas putida UW4 which expresses a high level of ACC deaminase and its ACC deaminase-minus mutant were compared to untreated seedlings along with a transgenic line of canola expressing the ACC deaminase enzyme in the roots. ACC deaminase breaks down 1-aminocyclopropane-1-carboxylic acid, the biosynthetic precursor to the plant hormone ethylene, lowering ethylene levels and improving plant fitness. Plants treated with the ACC deaminase-containing bacteria and transgenic plants expressing ACC deaminase are more tolerant to a variet of stresses and this expression study helps to illuminate the pathways responsible for the growth promotion provided by the beneficial bacteria and the role of the enzyme itself.

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Transcription profiling of canola trangenic plants expressing ACC deaminase and growth in the presence of nickel

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Accessing and Identification of Novel Environmental Alleles of the ACC Deaminase Domain Region through a Competition Assay, artificial and natural AccD competition constructs

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Project description:In this study, we set out to reprogram deaminase context specificity to pinpoint editing. We identified multiple nucleic acid-recognition hotspots in the E. coli tRNA-specific adenosine deaminase (TadA). Strategically sampling these recognition hotspots, we first accessed multipotency for C in TadA and subsequently eliminate its A-deamination activity. We further reprogrammed TadAC context specificity through 16 evolution campaigns, each aimed at a defined NCN context, and isolated hundreds of thousands of context-specific cytosine deaminases. Our panel of 16 NCN-specific deaminases covers the full spectrum of all possible minus1 and plus 1 contexts for a target C, offering on demand deaminase choices for editor customization. Our context-specific CBEs corrected 5,866 of 7,196 disease-associated T:A-to-C:G transitions documented by ClinVar with higher accuracy than existing CBEs, often achieving selective editing of a single cytosine out of multiple cytosines in the protospacer without compromising editing potency. We also showcased the application of context-specific base editing for modeling disease-associated C:G-to-T:A transitions using two cancer driver mutations, KRASG12D and TP53R248Q, each demanding selective editing of one cytosine in two consecutive cytosines (ACC and CCG). These context-specific editors, as expected, showed tightly controlled off-target profiles by rejecting most cytosines at potential off-target sites. Bystander-free, single-nucleobase editing, as enabled by reprogramming deaminase context specificity, complements our current editor portfolio and unlocks new potential in base editing.

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