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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Structural switching dynamically controls the doubly pseudoknotted Rous sarcoma virus–programmed ribosomal frameshifting element

A hallmark of retrovirus replication is the translation of two different polyproteins from one RNA through programmed –1 frameshifting. This is a mechanism in which the actively translating ribosome is induced to slip in the 5′ direction at a defined codon and then continues translating in the new reading frame. Programmed frameshifting controls the stoichiometry of viral proteins and is therefore under stringent evolutionary selection. Forty years ago, the first frameshifting stimulatory element was discovered in the Rous sarcoma virus. The ~120 nt RNA segment was predicted to contain a pseudoknot, but its 3D structure has remained elusive. Now, we have determined cryoEM and X-ray crystallographic structures of this classic retroviral element, finding that it adopts a butterfly-like double-pseudoknot fold. One “wing” contains a dynamic pyrimidine-rich helix, observed crystallographically in two conformations and in a third conformation via cryoEM. The other wing encompasses the predicted pseudoknot, which interacts with a second unexpected pseudoknot through a toggle residue, A2546. This key purine switches conformations between structural states and tunes the stability of interacting residues in the two wings. We find that its mutation can modulate frameshifting by as much as 50-fold, likely by altering the relative abundance of different structural states in the conformational ensemble of the RNA. Taken together, our structure–function analyses reveal how a dynamic double pseudoknot junction stimulates frameshifting by taking advantage of conformational heterogeneity, supporting a multistate model in which high Shannon entropy enhances frameshifting efficiency.

Science & Technology - Other Topics↗
DOE OSTI2025

Allosteric inhibition of the IZUMO1–JUNO fertilization complex by the naturally occurring antisperm antibody OBF13

Sperm IZUMO1 binds to egg JUNO, and this interaction is essential for mammalian fertilization. Isolated from a female mouse immunized with syngeneic sperm, the antisperm antibody OBF13 recognizes IZUMO1 and inhibits murine fertilization. How OBF13 interferes with sperm–egg interactions was unknown. Here, we present the X-ray crystal structure of IZUMO1 in complex with OBF13. OBF13 binds to the apex of the four-helix domain of IZUMO1, distant from the JUNO-binding site. Our crystal structure of OBF13-bound IZUMO1 resembles apo-IZUMO1 and differs from the structure of IZUMO1 in complex with JUNO. We identify that OBF13 carries a low level of somatic hypermutation, and through deep mutational scanning, we engineer an affinity-enhanced OBF13 variant. This OBF13 variant single-chain fragment variable decreases the apparent affinity of IZUMO1 for membrane-bound murine JUNO and blocks the binding of acrosome-reacted sperm to eggs, thereby preventing fertilization. We propose allostery between the OBF13 epitope and the JUNO-binding site. OBF13 inhibits a conformational change in IZUMO1, preventing fusion-competent sperm from adhering to murine eggs during fertilization. Surprisingly, murine IZUMO1 binds to hamster JUNO with an affinity ~20-fold higher than to murine JUNO. The decreased affinity caused by OBF13 of murine IZUMO1 for hamster JUNO is sufficient for murine sperm to bind to and fuse with hamster eggs. Our studies provide a structural and mechanistic framework for species-specific, allosteric inhibition of IZUMO1 by a naturally occurring antisperm antibody and offer insights into the development of immunocontraceptives.

Science & Technology - Other Topics↗
DOE OSTI2025

Factors underlying a latitudinal gradient in the S/G lignin monomer ratio in natural poplar variants

The chemical composition of wood plays a pivotal role in the adaptability and structural integrity of trees. However, few studies have investigated the environmental factors that determine lignin composition and its biological significance in plants. Here, we examined the lignin syringyl-to-guaiacyl (S/G) ratio in members of a Populus trichocarpa population sourced from their native habitat and conducted a genome wide association study to identify genes linked to lignin formation. Our results revealed many significant associations, suggesting that lignin biosynthesis is a complex polygenic trait. Additionally, we found an increase in the S/G ratio from northern to southern geographic origin of the trees sampled, along with a corresponding metabolic and transcriptional reprogramming of xylem cell wall biosynthesis. Further molecular analysis identified a mutation in a cell wall laccase genetically associated with higher S/G ratios that predominate in trees from warmer lower latitudes. Collectively, our findings suggest that lignin heterogeneity arises from an evolutionary process enabling poplar adaptation to different climatic challenges.

adaptation↗
DOE OSTI2025

An in vitro BRAF activation assay elucidates molecular mechanisms driving disassembly of the autoinhibited BRAF state

The RAF kinases (ARAF, BRAF, and CRAF) are essential components of the RAS-ERK signaling pathway, which controls vital cellular processes and is frequently dysregulated in human disease. Notably, mutations that alter BRAF function are prominent drivers of human cancer and certain RASopathy disorders, making BRAF an important target for therapeutic intervention. Despite extensive research, several aspects of BRAF regulation remain unclear. In this study, we developed an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-3 2 :MEK complexes. Our results show that fully processed, active-state KRAS alone can promote dimer-dependent BRAF activation. Moreover, we found that phosphatidylserine (PS)-containing liposomes synergized with KRAS to promote BRAF activation, achieving activity levels comparable to those observed with BRAF proteins that constitutively dimerize. In contrast, the SMP phosphatase complex had only a minimal effect on BRAF catalytic activity in this system but mediated the dephosphorylation of the negative regulatory pS365 14-3-3 binding site in a manner that was accelerated by the presence of KRAS alone or KRAS and 30% PS liposomes. Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential.

BRAF↗
DOE OSTI2024

Spatial Proteomics towards cellular Resolution

Introduction: Spatial biology is an emerging interdisciplinary field facilitating biological discoveries through the use of spatial omics technologies. Recent advancements in spatial transcriptomics, spatial genomics (e.g. genetic mutations and epigenetic marks), multiplexed immunofluorescence, and spatial metabolomics/lipidomics have enabled high-resolution spatial profiling of gene expression, genetic variation, protein expression, and metabolites/lipids profiles in tissue. These developments contribute to a deeper understanding of the spatial organization within tissue microenvironments at the molecular level. Areas covered: This report provides an overview of the untargeted, bottom-up mass spectrometry (MS)-based spatial proteomics workflow. It highlights recent progress in tissue dissection, sample processing, bioinformatics, and liquid chromatography (LC)-MS technologies that are advancing spatial proteomics toward cellular resolution. Expert opinion: The field of untargeted MS-based spatial proteomics is rapidly evolving and holds great promise. To fully realize the potential of spatial proteomics, it is critical to advance data analysis and develop automated and intelligent tissue dissection at the cellular or subcellular level, along with high-throughput LC-MS analyses of thousands of samples. In conclusion, achieving these goals will necessitate significant advancements in tissue dissection technologies, LC-MS instrumentation, and computational tools.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Investigating the mechanism of chloroplast singlet oxygen signaling in the Arabidopsis thaliana accelerated cell death 2 mutant

As sessile organisms, plants have evolved complex signaling mechanisms to sense stress and acclimate. This includes the use of reactive oxygen species (ROS) generated during dysfunctional photosynthesis to initiate signaling. One such ROS, singlet oxygen ( 1 O 2 ), can trigger retrograde signaling, chloroplast degradation, and programmed cell death. However, the signaling mechanisms are largely unknown. Several proteins (e.g. PUB4, OXI1, EX1) are proposed to play signaling roles across three Arabidopsis thaliana mutants that conditionally accumulate chloroplast 1 O 2 (fluorescent in blue light (flu), chlorina 1 (ch1), and plastid ferrochelatase 2 (fc2)). We previously demonstrated that these mutants reveal at least two chloroplast 1 O 2 signaling pathways (represented by flu and fc2/ch1). Here, we test if the 1 O 2 -accumulating lesion mimic mutant, accelerated cell death 2 (acd2), also utilizes these pathways. The pub4–6 allele delayed lesion formation in acd2 and restored photosynthetic efficiency and biomass. Conversely, an oxi1 mutation had no measurable effect on these phenotypes. acd2 mutants were not sensitive to excess light (EL) stress, yet pub4–6 and oxi1 both conferred EL tolerance within the acd2 background, suggesting that EL-induced 1 O 2 signaling pathways are independent from spontaneous lesion formation. Thus, 1 O 2 signaling in acd2 may represent a third (partially overlapping) pathway to control cellular degradation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Mechanisms of a novel regulatory light chain–dependent cardiac myosin inhibitor

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the heart characterized by thickening of the left ventricle (LV), hypercontractility, and impaired relaxation. HCM is caused primarily by heritable mutations in sarcomeric proteins, such as β myosin heavy chain. Until recently, medications in clinical use for HCM did not directly target the underlying contractile changes in the sarcomere. Here, we investigate a novel small molecule, RLC-1, identified in a bovine cardiac myofibril high-throughput screen. RLC-1 is highly dependent on the presence of a regulatory light chain to bind to cardiac myosin and modulate its ATPase activity. In demembranated rat LV trabeculae, RLC-1 decreased maximal Ca2+-activated force and Ca2+ sensitivity of force, while it increased the submaximal rate constant for tension redevelopment. In myofibrils isolated from rat LV, both maximal and submaximal Ca2+-activated force are reduced by nearly 50%. Additionally, the fast and slow phases of relaxation were approximately twice as fast as DMSO controls, and the duration of the slow phase was shorter. Structurally, x-ray diffraction studies showed that RLC-1 moved myosin heads away from the thick filament backbone and decreased the order of myosin heads, which is different from other myosin inhibitors. In intact trabeculae and isolated cardiomyocytes, RLC-1 treatment resulted in decreased peak twitch magnitude and faster activation and relaxation kinetics. In conclusion, RLC-1 accelerated kinetics and decreased force production in the demembranated tissue, intact tissue, and intact whole cells, resulting in a smaller cardiac twitch, which could improve the underlying contractile changes associated with HCM.

Physiology↗
DOE OSTI2024

Calcium has a direct effect on thick filament activation in porcine myocardium

Sarcomere activation in striated muscle requires both thin filament–based and thick filament–based activation mechanisms. Recent studies have shown that myosin heads on the thick filaments undergo OFF to ON structural transitions in response to calcium (Ca2+) in permeabilized porcine myocardium in the presence of a small molecule inhibitor that eliminated active force. The changes in X-ray diffraction signatures of OFF to ON transitions were interpreted as Ca2+ acting to activate the thick filaments. Alternatively, Ca2+ binding to troponin could initiate a Ca2+-dependent crosstalk from the thin filament to the thick filament via interfilament connections such as the myosin binding protein-C. Here, we exchanged native troponin in permeabilized porcine myocardium for troponin containing the cTnC D65A mutation, which disallows the activation of troponin through Ca2+ binding to determine if Ca2+-dependent thick filament activation persists in the absence of thin filament activation. After the exchange protocol, over 95% of the Ca2+-activated force was eliminated. Equatorial intensity ratio increased significantly in both WT and D65A exchanged myocardium with increasing Ca2+ concentration. The degree of helical ordering of the myosin heads decreased by the same amount in WT and D65A myocardium when Ca2+ concentration increased. These results are consistent with a direct effect of Ca2+ in activating the thick filament rather than an indirect effect due to Ca2+-mediated crosstalk between the thick and thin filaments.

Physiology↗
DOE OSTI2024

ULTRA-effective labeling of tandem repeats in genomic sequence

In the age of long read sequencing, genomics researchers now have access to accurate repetitive DNA sequence (including satellites) that, due to the limitations of short read-sequencing, could previously be observed only as unmappable fragments. Tools that annotate repetitive sequence are now more important than ever, so that we can better understand newly uncovered repetitive sequences, and also so that we can mitigate errors in bioinformatic software caused by those repetitive sequences. To that end, we introduce the 1.0 release of our tool for identifying and annotating locally repetitive sequence, ULTRA Locates Tandemly Repetitive Areas (ULTRA). ULTRA is fast enough to use as part of an efficient annotation pipeline, produces state-of-the-art reliable coverage of repetitive regions containing many mutations, and provides interpretable statistics and labels for repetitive regions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The genome of the polyextremophilic yeast, Naganishia friedmannii, reveals adaptations involved in stress response pathways, carbohydrate metabolism expansion, and a limited DNA repair repertoire

Here we report the draft genome sequence of Naganishia friedmannii (formerly Cryptococcus friedmannii) isolate, a Basidiomycota yeast commonly found in some of the most extreme environments of the Earth's cryosphere. We isolated N. friedmannii strain Llullensis from soils at 6000 m above sea level on Volcán Llullaillaco, Argentina. The genome was 22.2 Mb with 6251 identified protein coding genes. Proteins known to be associated with thermal, osmotic, and radiation stress were identified in the genome. Comparative analysis with seven other Naganishia genomes revealed unique features underlying its polyextremophilic lifestyle. Naganishia friedmannii showed an expansion of genes involved in breaking down plant-derived carbohydrates, supporting the hypothesis that it survives at high elevations by metabolizing wind-deposited organic matter. Surprisingly, many genes involved in cell-cycle checkpoints and DNA repair were missing, as in several other Naganishia species. This extensive loss may be adaptive in extreme environments prone to abiotic stress, where a high mutation rate could generate advantageous traits, and reduced cell-cycle control may allow for faster reproduction that would be advantageous for rapid growth during brief periods of soil wetting following rare snow events.

Vimercati, Lara↗
DOE OSTI2025

Targeted seed EMS mutagenesis reveals a basic helix–loop–helix transcription factor underlying male sterility in sorghum

Abstract Forward genetic screens of mutant populations are fundamental for functional genomics studies. However, isolating independent mutant alleles to molecularly identify causal genes is challenging in species recalcitrant to genetic manipulation. Here, we demonstrate that classic seed ethyl methanesulfonate (EMS) mutagenesis coupled with genome sequencing can overcome this limitation in sorghum. We used this method to generate new mutant alleles of sorghum MALE STERILE 8 (MS8) and identified the causal locus for the ms8 phenotype as Sobic.004G270900, which encodes the sorghum ortholog of maize bhlh122, a basic helix–loop–helix (bHLH) transcription factor required for male fertility in maize. Bulked segregant analysis mapped ms8-1 to a region on chromosome 4 containing Sobic.004G270900. Seeds from heterozygous MS8/ms8-1 plants were mutagenized and screened for chimeric inflorescences containing sectors with white, sterile anthers resembling the ms8-1 homozygous phenotype. DNA sequencing of sterile and fertile sectors from a single chimeric inflorescence revealed two mutations in Sobic.004G270900 within the sterile sector, but not the fertile sector. Isolation of this loss-of-function allele (ms8-2) established Sobic.004G270900 as the causative locus for male sterility in the ms8 mutant. We generated additional alleles of MS8 in a different genetic background using CRISPR/Cas9-based gene editing, where deletions in Sobic.004G270900 also resulted in male sterility. Our work identified a gene underlying male sterility in sorghum and provides a novel and straightforward genetic tool for researchers who lack access to advanced transformation facilities to validate gene candidates. Unlike gene editing, no prior knowledge of candidate genes is required for targeted seed EMS mutagenesis to aid identification of causal loci.

Genetics & Heredity↗
DOE OSTI2025

Artemether-Lumefantrine Treatment Selects Plasmodium falciparum Multidrug Resistance 1 ( pfmdr1 ) Increased Copy Number Among African Malaria Infections

Abstract Background Decreased efficacy of artemether-lumefantrine, the globally most used antimalarial, has recently emerged in Africa. Methods An efficacy trial was carried out based on directly observed artemether-lumefantrine therapy at Bengo, Northern Angola. One-hundred Plasmodium falciparum uncomplicated malaria patients (2–10 years old) were enrolled, hospitalized for the treatment period, and followed up for 42 days. Polymerase chain reaction (PCR) correction was performed with pfmsp1/2 plus glurp, with analysis considering 2 or 3 coincident markers. Infections were tested by quantitative PCR (qPCR) for pfmdr1 copy number (pfmdr1×N), a potential P. falciparum marker of lumefantrine resistance previously identified in the region. In vitro clone mixtures were built and used to determine the relation between qPCR copy number scores and actual intrainfection quantitative fractions of pfmdr1×N. Results We observed a significant posttreatment selection of gene amplification, suggesting a role in the parasite in vivo response to this drug. pfmdr1×2 qPCR scores of 1.3, 1.4, and 1.5 were determined to correspond to 15%, 25%, and 35% intrainfection rates. Patients carrying infections with a score ≥1.4 at baseline were linked to decreased artemether-lumefantrine day 42 efficacy (79% vs 97% single-copy pfmdr1). All infections were pfmdr1 N86 carriers and no pfk13 mutations were found. Conclusions Our study suggests pfmdr1×N as a marker of P. falciparum in vivo response to lumefantrine in Africa, while indicating patients carrying infections with a pretreatment pfmdr1×N score ≥1.4 before treatment are a group experiencing decreased artemether-lumefantrine performance.

Fançony, Claudia (ORCID:0000000344211769)↗
DOE OSTI2024

Distinct function of Chlamydomonas CTRA-CTR transporters in Cu assimilation and intracellular mobilization

Abstract Successful acclimation to copper (Cu) deficiency involves a fine balance between Cu import and export. In the green alga Chlamydomonas reinhardtii, Cu import is dependent on a transcription factor, Copper Response Regulator 1 (CRR1), responsible for activating genes in Cu-deficient cells. Among CRR1 target genes are two Cu transporters belonging to the CTR/COPT gene family (CTR1 and CTR2) and a related soluble protein (CTR3). The ancestor of these green algal proteins was likely acquired from an ancient chytrid and contained conserved cysteine-rich domains (named the CTR-associated domains, CTRA) that are predicted to be involved in Cu acquisition. We show by reverse genetics that Chlamydomonas CTR1 and CTR2 are canonical Cu importers albeit with distinct affinities, while loss of CTR3 did not result in an observable phenotype under the conditions tested. Mutation of CTR1, but not CTR2, recapitulates the poor growth of crr1 in Cu-deficient medium, consistent with a dominant role for CTR1 in high-affinity Cu(I) uptake. On the other hand, the overaccumulation of Cu(I) (20 times the quota) in zinc (Zn) deficiency depends on CRR1 and both CTR1 and CTR2. CRR1-dependent activation of CTR gene expression needed for Cu over-accumulation can be bypassed by the provision of excess Cu in the growth medium. Over-accumulated Cu is sequestered into the acidocalcisome but can become remobilized by restoring Zn nutrition. This mobilization is also CRR1-dependent, and requires activation of CTR2 expression, again distinguishing CTR2 from CTR1 and consistent with the lower substrate affinity of CTR2. One sentence summary Regulation of Cu uptake and sequestration by members of the CTR family of proteins in Chlamydomonas.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar K D

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer–ligand complex with a remarkable set of properties: Its unprecedented K D of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structures of the mitochondrial single-stranded DNA binding protein with DNA and DNA polymerase γ

Abstract The mitochondrial single-stranded DNA (ssDNA) binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA that could impede downstream replication or repair processes. Clinical mutations in the SSBP1 gene have been linked to a range of mitochondrial disorders affecting nearly all organs and systems. Yet, the molecular determinants governing the interaction between mtSSB and ssDNA have remained elusive. Similarly, the structural interaction between mtSSB and other replisome components, such as the mitochondrial DNA polymerase, Polγ, has been minimally explored. Here, we determined a 1.9-Å X-ray crystallography structure of the human mtSSB bound to ssDNA. This structure uncovered two distinct DNA binding sites, a low-affinity site and a high-affinity site, confirmed through site-directed mutagenesis. The high-affinity binding site encompasses a clinically relevant residue, R38, and a highly conserved DNA base stacking residue, W84. Employing cryo-electron microscopy, we confirmed the tetrameric assembly in solution and capture its interaction with Polγ. Finally, we derived a model depicting modes of ssDNA wrapping around mtSSB and a region within Polγ that mtSSB binds.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Self-assembly and condensation of intermolecular poly(UG) RNA quadruplexes

Abstract Poly(UG) or ‘pUG’ dinucleotide repeats are highly abundant sequences in eukaryotic RNAs. In Caenorhabditis elegans, pUGs are added to RNA 3′ ends to direct gene silencing within Mutator foci, a germ granule condensate. Here, we show that pUG RNAs efficiently self-assemble into gel condensates through quadruplex (G4) interactions. Short pUG sequences form right-handed intermolecular G4s (pUG G4s), while longer pUGs form left-handed intramolecular G4s (pUG folds). We determined a 1.05 Å crystal structure of an intermolecular pUG G4, which reveals an eight stranded G4 dimer involving 48 nucleotides, 7 different G and U quartet conformations, 7 coordinated potassium ions, 8 sodium ions and a buried water molecule. A comparison of the intermolecular pUG G4 and intramolecular pUG fold structures provides insights into the molecular basis for G4 handedness and illustrates how a simple dinucleotide repeat sequence can form complex structures with diverse topologies.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Crystal structure of MutYX: a novel clusterless adenine DNA glycosylase with a distinct C-terminal domain and 8-oxoguanine recognition sphere

Abstract The [4Fe–4S] cluster is an important cofactor of the base excision repair (BER) adenine DNA glycosylase MutY to prevent mutations associated with 8-oxoguanine (OG). Several MutYs lacking the [4Fe–4S] cofactor have been identified. Phylogenetic analysis shows that clusterless MutYs are distributed in two clades suggesting cofactor loss has occurred in multiple independent evolutionary events. Herein, we determined the first crystal structure of a clusterless MutY complexed with DNA. On the basis of the dramatic structural divergence from canonical MutYs, we refer to this as representative of a clusterless MutY subgroup “MutYX.” Interestingly, MutYX compensates for the missing [4Fe–4S] cofactor to maintain positioning of catalytic residues by expanding a pre-existing α-helix and acquisition of a new α-helix. Surprisingly, MutYX also acquired a new C-terminal domain that uniquely recognizes OG using residues Gln201 and Arg209. Adenine glycosylase assays and binding affinity measurements indicate that Arg209 is the primary residue responsible for OG:A lesion specificity, while Gln201 assists by bridging OG and Arg209. Surprisingly, replacement of Arg209 and Gln201 with Ala increased activity toward G:A mismatches. The MutYX structure serves as an example of devolution, capturing structural features required to retain function in the absence of a metal cofactor considered indispensable.

Trasviña-Arenas, Carlos H [University of Californi↗
DOE OSTI2026

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli

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 also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.

Kammerdiener, Elise K. [Oak Ridge National Laborat↗