Biology subjects

Achmedov, T.

Publications and source records attributed to Achmedov, T..

4 recordsLinked to original sources

bioRxiv2024

Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here, we harness the bacterial anti-phage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives efficient homologous recombination in tested bacteria, offering flexible and scar-free genome editing without base replacement nor counterselection. In tested eukaryotes including yeast, plants and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of a chemical moiety to DNA to expand current modalities for precision gene editing.

synthetic biology↗

bioRxiv2024

AcrVIB1 inhibits CRISPR-Cas13b immunity by promoting unproductive crRNA binding accessible to RNase attack

Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immune defenses, with almost all known Acrs acting on the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex. Here, we show that AcrVIB1, the only known Acr against Cas13b, principally acts upstream of RNP complex formation by promoting unproductive crRNA binding followed by crRNA degradation. AcrVIB1 tightly binds to Cas13b but not to the Cas13b-crRNA complex, resulting in enhanced rather than blocked crRNA binding. However, the more tightly-bound crRNA does not undergo processing and exhibits altered target RNA binding that fails to activate collateral RNA cleavage. The bound crRNA is also accessible to RNases, leading to crRNA turnover in vivo even in the presence of Cas13b. Finally, cryo-EM structures revealed that AcrVIB1 binds a helical domain of Cas13b responsible for securing the crRNA, keeping the domain in an untethered state. These findings reveal an Acr that converts an effector nuclease into a crRNA sink to suppress CRISPR-Cas defense. HighlightsO_LIAcrVIB1 binds Cas13b in the absence of a crRNA C_LIO_LIThe bound AcrVIB1 promotes crRNA binding to Cas13b C_LIO_LIThe crRNA binds Cas13b unproductively and is accessible to RNases C_LIO_LIAcrVIB1 binds the Helical-2 domain of Cas13b, preventing it from securing the crRNA C_LI

biochemistry↗

bioRxiv2022

Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria

Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca. We further applied atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae, establishing a new resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.

synthetic biology↗

bioRxiv2021

Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from {sigma}70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the {Delta}recBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

synthetic biology↗