Search NASA⌕ Search

SEARCH · Search NASA

Results for “Cas9 engineering”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

69 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Knocking out barriers to engineered cell activity

Engineered T cell therapies are revolutionizing cancer treatment by achieving long-lasting remission in blood-related cancers, such as leukemia and lymphoma. These therapies involve removal of patient T cells, “reprogramming” them to attack cancer cells, and then transferring them back into the patient. Targeted gene inactivation (knockout) using CRISPR-Cas9 can enhance T cell activity (1, 2) and has the potential to expand cell therapy applications. Until now, it has been unknown whether CRISPR-Cas9–edited T cells would be tolerated and thrive once reinfused into a human. On page 1001 of this issue, Stadtmauer et al. (3) present data from a phase 1 clinical trial (designed to test safety and feasibility) on the first cancer patients treated with CRISPR-Cas9–modified T cells. The findings represent an important advance in the therapeutic application of gene editing and highlight the potential to accelerate development of cell-based therapies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

CRISPR/Cas9-based gene activation and base editing in Populus

The genus Populus has long been used for environmental, agroforestry and industrial applications worldwide. Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological research. As such, various modern biotechnologies, including CRISPR/Cas9-based techniques, have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin composition. However, CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone “717-1B4” (P. tremula x P. alba clone INRA 717-1B4). Alternative CRISPR/Cas9-based technologies, e.g. those involving modified Cas9 for gene activation and base editing, have not been evaluated in most Populus species for their efficacy. Here we employed a deactivated Cas9 (dCas9)-based CRISPR activation (CRISPRa) technique to fine-tune the expression of two target genes, TPX2 and LecRLK-G which play important roles in plant growth and defense response, in hybrid poplar clone “717-1B4” and poplar clone “WV94” (P. deltoides “WV94”), respectively. We observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation, demonstrating the effectiveness of dCas9-based CRISPRa system in Populus. In addition, we applied Cas9 nickase (nCas9)-based cytosine base editor (CBE) to precisely introduce premature stop codons via C-to-T conversion, with an efficiency of 13%–14%, in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in hybrid poplar clone “717-1B4”. Overall, we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species, facilitating the adoption of emerging genome editing tools in woody species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens

Abstract High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica and acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Progress Towards a New Model Chemolithoautotrophic Host (CRADA Final Report)

Some of the largest scale chemical processes for the production of important commodity chemicals and fuels involve an H 2 -containing gas mixture as an intermediate [e.g. ammonia, urea, methanol, Fischer-Tropsch (F-T) diesel, and hydrotreatment of crude petroleum to refined fuels]. The successful development of a model H 2 -oxidizing chemoautotrophic host could expand the range of fuels and chemicals produced from H 2 and CO 2 intermediates in the chemical, oil, and gas sectors in the near term, as well as from renewable and waste-derived sources of these gases that are expected to greatly expand in coming years. Among non-photosynthetic bacteria that can utilize H 2 and CO 2 , Cupriavidus necator (C. necator, formerly Ralstonia eutropha), is the best studied. C. necator is an excellent microbial host for the production of a variety of chemicals because it grows extremely quickly to very high cell densities autotrophically on H 2 and CO 2 , is genetically tractable, and has the ability to accumulate polymers, such as polyhydroxybutyrate, at industrial levels. Despite having great potential as a platform bioproduction host, genetic tools are limited, making metabolic engineering of this organism slow and laborious. In this CRADA project, we developed a number of genetic tools for C. necator: 1) Improvement of C. necator genetic transformation efficiency 2) Integration of heterologous genes into the C. necator chromosome and development of promoter library 3) Development of graded RBSs to control heterologous protein expression 4) Use of RBSs to demonstrate fatty alcohol production in C. necator 5) Demonstration of CRISPR-Cas9 gene editing in C. necator

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes

The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. Furthermore, this work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: Azotobacter vinelandii, Stutzerimonas stutzeri (Pseudomonas stutzeri), and a new isolate of Klebsiella variicola. All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. S. stutzeri and K. variicola showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440

Summary The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. A number of inducible promoters (P lac , P tac , P tetR/tetA and P bad ) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Expanding the genetic toolkit: adenine and cytosine base editors for gene disruption in Aspergillus niger

Despite revolutionizing fungal genetic engineering, conventional CRISPR/Cas9-mediated knockouts rely on DNA double-strand breaks (DSBs), which can cause unwanted insertions and deletions, chromosomal abnormalities, and cytotoxicity. Base editors such as adenine base editors (ABEs), which convert A‧T to G‧C, and cytosine base editors (CBEs), which convert C‧G to T‧A, offer a safer alternative by enabling predictable, target-specific single-nucleotide changes without introducing DSBs. To overcome the limitations of traditional genome editing in filamentous fungi, we developed efficient base-editing systems in Aspergillus niger . For the first time, we constructed an ABE in A. niger , achieving up to 80% editing efficiency and inducing predictable A-to-G mutations at the intended intron sites, disrupting gene function through mRNA mis-splicing. We also developed a highly efficient CBE system, capable of introducing premature stop codons with 50–100% efficiency. To broaden the editing scope, we implemented a Cas9-NG variant recognizing a relaxed PAM sequence requiring only a single guanine (G), enabling editing at start codons and splice sites. Leveraging this expanded scope, we established gene disruption approaches by targeting start codons via ABE-mediated A-to-G conversions (ATG-to-GTG and ATG-to-ACG) and CBE-mediated C-to-T conversion (ATG-to-ATA). Additionally, our base-editing systems enable multiplex gRNA delivery and marker-free editing of multiple genes. Collectively, the scope-expanding strategies increase the number of genes targetable for disruption by base-editing in A. niger by 26.3% and enable near-complete coverage of 96% of the coding genes. Overall, this work demonstrates the potential of ABE and CBE systems as versatile, efficient, and safer alternatives to DSBs-based gene disruption in filamentous fungi.

Aspergillus↗
DOE OSTI2024

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Knocking out the carboxyltransferase interactor 1 (CTI1) in Chlamydomonas boosted oil content by fivefold without affecting cell growth

Summary The first step in chloroplast de novo fatty acid synthesis is catalysed by acetyl‐CoA carboxylase (ACCase). As the rate‐limiting step for this pathway, ACCase is subject to both positive and negative regulation. In this study, we identify a Chlamydomonas homologue of the plant carboxyltransferase interactor 1 (CrCTI1) and show that this protein interacts with the Chlamydomonas α‐carboxyltransferase (Crα‐CT) subunit of the ACCase by yeast two‐hybrid protein–protein interaction assay. Three independent CRISPR‐Cas9 mediated knockout mutants for CrCTI1 each produced an ‘enhanced oil’ phenotype, accumulating 25% more total fatty acids and storing up to fivefold more triacylglycerols (TAGs) in lipid droplets. The TAG phenotype of the crcti1 mutants was not influenced by light but was affected by trophic growth conditions. By growing cells under heterotrophic conditions, we observed a crucial function of CrCTI1 in balancing lipid accumulation and cell growth. Mutating a previously mapped in vivo phosphorylation site (CrCTI1 Ser108 to either Ala or to Asp), did not affect the interaction with Crα‐CT. However, mutating all six predicted phosphorylation sites within Crα‐CT to create a phosphomimetic mutant reduced this pairwise interaction significantly. Comparative proteomic analyses of the crcti1 mutants and WT suggested a role for CrCTI1 in regulating carbon flux by coordinating carbon metabolism, antioxidant and fatty acid β‐oxidation pathways, to enable cells to adapt to carbon availability. Taken together, this study identifies CrCTI1 as a negative regulator of fatty acid synthesis in algae and provides a new molecular brick for the genetic engineering of microalgae for biotechnology purposes.

Li, Zhongze [Aix‐Marseille Université, CEA, CNRS, ↗
DOE OSTI2022

In vivo Functional Characterization of Hydrophilic X2 Modules in the Cellulosomal Scaffolding Protein

As part of free cellulases or scaffolding proteins in cellulosomes, the hydrophilic non-catalytic X2 module is widely distributed in cellulolytic Clostridia or other Firmicutes bacteria. Previous biochemical studies suggest that X2 modules might increase the solubility and substrate binding affinity of X2-bearing proteins. However, their in vivo biological functions remain elusive. Here we employed CRISPR-Cas9 editing to genetically modify X2 modules by deleting the conserved motif (NGNT) from the CipC scaffoldin. Both single and double X2 mutants (X2-N: near the N terminus of CipC; X2-C: near the C terminus of CipC) presented similar stoichiometric compositions in isolated cellulosomes as the wildtype strain (WT). These X2 mutants had an elongated adaptation stage during growth on cellulose compared to cellobiose. Compared to WT, the double mutant ΔX2-NC reduced cellulose degradation by 15% and the amount of released soluble sugars by 63%. Since single X2 mutants did not present such obvious physiological changes as ΔX2-NC, there seems to be a functional redundancy between X2 modules in CipC. The in vivo adhesion assay revealed that ΔX2-NC decreased cell attachment to cellulose by 70% but a weaker effect was also overserved in single X2 mutants. These results highlight the in vivo biological role of X2 in increasing cellulose degradation efficiency by enhancing the binding affinity between cells and cellulose, which provides new perspectives for microbial engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria

With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’

Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood (Populus deltoides) clone WV94 can be transformed by A. tumefaciens, but several challenges remain unresolved, including the relatively low transformation efficiency and the relatively high rate of false positives from antibiotic-based selection of transgenic events. Moreover, the efficacy of CRISPR-Cas system has not been explored in P. deltoides yet. Here, we first optimized the protocol for Agrobacterium-mediated stable transformation in P. deltoides WV94 and applied a UV-visible reporter called eYGFPuv in transformation. Our results showed that the transgenic events in the early stage of transformation could be easily recognized and counted in a non-invasive manner to narrow down the number of regenerated shoots for further molecular characterization (at the DNA or mRNA level) using PCR. We found that approximately 8.7% of explants regenerated transgenic shoots with green fluorescence within two months. Next, we examined the efficacy of multiplex CRISPR-based genome editing in the protoplasts derived from P. deltoides WV94 and hybrid poplar clone ‘52-225’ (P. trichocarpa × P. deltoides clone ‘52-225’). The two constructs expressing the Trex2-Cas9 system resulted in mutation efficiency ranging from 31% to 57% in hybrid poplar clone 52-225, but no editing events were observed in P. deltoides WV94 transient assay. The eYGFPuv-assisted plant transformation and genome editing approach demonstrated in this study has great potential for accelerating the genome editing-based breeding process in poplar and other non-model plants species and point to the need for additional CRISPR work in P. deltoides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cas9 deletion of lutein biosynthesis in the marine alga Picochlorum celeri reduces photosynthetic pigments while sustaining high biomass productivity

Domestication of algae for food and renewable biofuels remains limited by the low photosynthetic efficiencies of processes that have evolved to be competitive for optimal light capture, incentivizing the development of large antennas in light-limiting conditions, thus decreasing efficient light utilization in cultivated ponds or photobioreactors. Reducing the pigment content to improve biomass productivity has been a strategy discussed for several decades and the ability to reduce pigment significantly is now fully at hand thanks to the widespread use of genome editing tools. Picochlorum celeri is one of the fastest growing marine algae identified and holds particular promise for outdoor cultivation, especially in saline water and warm climates. We show that while chlorophyll b is essential to sustain high biomass productivities under dense cultivation, removing Picochlorum celeri’s main carotenoid, lutein, leads to a decreased total chlorophyll content, higher a/ b ratio, reduced functional LHCII cross section and higher maximum quantum efficiencies at lower light intensities, resulting in an incremental increase in biomass productivity and increased PAR-to-biomass conversion efficiency. These findings further strengthen the existing strategies to improve photosynthetic efficiency and biomass production in algae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A High-efficacy CRISPRi System for Gene Function Discovery in Zymomonas mobilis

Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.

lignocellulosic hydrolysate↗