Biology subjects

Eckert, C. A.

Publications and source records attributed to Eckert, C. A..

6 recordsLinked to original sources

bioRxiv2025

When good guides go bad: empirical evaluation of all unique Cas9 targets in E. coli reveal widespread functionality and rules for gRNA biological design

The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease in which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption that inactive gRNAs are rare persists in the E. coli community. To resolve these contradictions, we evaluated the activity of nearly 500,000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional.These nonfunctional gRNAs exhibit strong spacer self-interaction, which can be either excluded using a simple design rule or "repaired" during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million E. coli gRNAs that have been empirically evaluated in vivo as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/651106v3_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1038ddcorg.highwire.dtl.DTLVardef@566e37org.highwire.dtl.DTLVardef@5df6b2org.highwire.dtl.DTLVardef@ef453c_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs (gRNAs). This information should be applicable to all Cas9-based methods and technologies. NoticeThis manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (https://www.energy.gov/doe-public-access-plan). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/651296v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1cb97a4org.highwire.dtl.DTLVardef@150bf11org.highwire.dtl.DTLVardef@2ffefforg.highwire.dtl.DTLVardef@10e24c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Ribozyme-based biosensor for imaging gene expression in plants

Detection of gene expression in plants is critical for understanding the molecular basis of complex plant biosystems and plant responses to environmental stresses. Here, we report the development of a split ribozyme-based biosensor that enables in vivo visualization of gene expression in plants. We demonstrated the utility of this biosensor in transient expression experiments (i.e., leaf infiltration in Nicotiana benthamiana) to detect RNAs derived from transgenes and tobacco rattle virus, respectively. Furthermore, we successfully engineered a split ribozyme-based biosensor in Arabidopsis thaliana for in vivo visualization of endogenous gene expression at the cellular level. In addition, we developed a platform for easy incorporation of different reporters into the RNA biosensor.

synthetic biology↗

bioRxiv2024

High-efficiency transformation and gene expression in Picosynechococcus sp. PCC 7002

Cyanobacteria are promising microbial platforms for a myriad of biotechnological applications, from sustainable biomaterials to photosynthetic chemical production, but still lack the breadth of genetic tools available for more commonly engineered microbes such as Escherichia coli. This study develops genetic tools to enhance the transformation efficiency and heterologous gene expression in Picosynechococcus sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain. Integration of fluorescent reporter cassettes across the genome revealed an integration site that yields a fourfold improvement in gene expression relative to previously reported sites. Protocol optimization and engineered DNA methylation in E. coli increased transformation efficiency by over tenfold. This work provides an experimental framework for efficient genome editing and metabolic engineering in the model cyanobacterium PCC 7002.

synthetic biology↗

bioRxiv2024

Discovering methylated DNA motifs in bacterial nanopore sequencing data with MIJAMP

Bacterial DNA methylation is involved in diverse cellular functions ranging from modulation of gene expression, DNA repair, and restriction-modification systems for defense against viruses and other foreign DNA. Methylome analysis determines sites within a bacterial chromosome that are methylated, revealing motifs that may be targeted by native restriction enzymes. Identification of these motifs is therefore critical to making an organism genetically tractable, where mimicking the methylome patterns in Escherichia coli allows plasmid DNA to be protected from restriction in the target organism and can therefore drastically enhance transformation efficiency. Oxford Nanopore Technologies (ONT) sequencing can detect methylated bases during sequencing, but software is needed to identify the corresponding methylated motifs within the data. Here, we develop MIJAMP (MIJAMP Is Just A MethylBED Parser), a software package that was developed to discover methylated motifs from the output of ONTs Modkit or other data in the methylBED format. MIJAMP employs a human-driven refinement strategy that empirically validates all motifs against genome-wide methylation data, thus eliminating false, under-, or overexplained motifs. MIJAMP can also report methylation data on a specific, user-defined motif. Using MIJAMP, we determined the methylated motifs both in a control strain (wild type E. coli) and in Picosynecococcus sp. strain PCC7002, laying the foundation for improved transformation in this organism. MIJAMP is available at https://code.ornl.gov/alexander-public/mijamp/.

molecular biology↗

bioRxiv2022

An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation, with a concomitant decrease in the need for antibiotics. In turn, reduced overgrowth allowed longer co-cultivation periods, with a trend towards higher transformation frequencies for switchgrass, but not for rice.

plant biology↗