SEARCH · Search NASA

Results for “multiplex genome editing”

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.

SourceResearch connectionFrom yearThrough year

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

At least 19 records

All sources NASA NTRS DOE OSTI

DOE OSTI2024

CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans

Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. Here, we envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.

60 APPLIED LIFE 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 OSTI2025

Efficient mutagenesis and genotyping of maize inbreds using biolistics, multiplex CRISPR/Cas9 editing, and Indel-Selective PCR

CRISPR/Cas9 based genome editing has advanced our understanding of a myriad of important biological phenomena. Important challenges to multiplex genome editing in maize include assembly of large complex DNA constructs, few genotypes with efficient transformation systems, and costly/labor-intensive genotyping methods. Here we present an approach for multiplex CRISPR/Cas9 genome editing system that delivers a single compact DNA construct via biolistics to Type I embryogenic calli, followed by a novel efficient genotyping assay to identify desirable editing outcomes. We first demonstrate the creation of heritable mutations at multiple target sites within the same gene. Next, we successfully created individual and stacked mutations for multiple members of a gene family. Genome sequencing found off-target mutations are rare. Multiplex genome editing was achieved for both the highly transformable inbred line H99 and Illinois Low Protein1 (ILP1), a genotype where transformation has not previously been reported. In addition to screening transformation events for deletion alleles by PCR, we also designed PCR assays that selectively amplify deletion or insertion of a single nucleotide, the most common outcome from DNA repair of CRISPR/Cas9 breaks by non-homologous end-joining. The Indel-Selective PCR (IS-PCR) method enabled rapid tracking of multiple edited alleles in progeny populations. The ‘end to end’ pipeline presented here for multiplexed CRISPR/Cas9 mutagenesis can be applied to accelerate maize functional genomics in a broader diversity of genetic backgrounds.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Data for "Efficient Mutagenesis and Genotyping of Maize Inbreds Using Biolistics, Multiplex CRISPR/Cas9 Editing, and Indel-Selective PCR"

gene editing↗

DOE OSTI2023

Unleashing the capacity of Rhodococcus for converting lignin into lipids

Bioconversion of bioresources/wastes (e.g., lignin, chemical pulping byproducts) represents a promising approach for developing a bioeconomy to help address growing energy and materials demands. Rhodococcus, a promising microbial strain, utilizes numerous carbon sources to produce lipids, which are precursors for synthesizing biodiesel and aviation fuels. However, compared to chemical conversion, bioconversion involves living cells, which is a more complex system that needs further understanding and upgrading. Various wastes amenable to bioconversion are reviewed herein to highlight the potential of Rhodococci for producing lipid-derived bioproducts. In light of the abundant availability of these substrates, Rhodococcus’ metabolic pathways converting them to lipids are analyzed from a “beginning-to-end” view. Based on an in-depth understanding of microbial metabolic routes, genetic modifications of Rhodococcus by employing emerging tools (e.g., multiplex genome editing, biosensors, and genome-scale metabolic models) are presented for promoting the bioconversion. Co-solvent enhanced lignocellulose fractionation (CELF) strategy facilitates the generation of a lignin-derived aromatic stream suitable for the Rhodococcus’ utilization. Novel alkali sterilization (AS) and elimination of thermal sterilization (ETS) approaches can significantly enhance the bioaccessibility of lignin and its derived aromatics in aqueous fermentation media, which promotes lipid titer significantly. In order to achieve value-added utilization of lignin, biodiesel and aviation fuel synthesis from lignin and lipids are further discussed. In conclusion, the possible directions for unleashing the capacity of Rhodococcus through synergistically modifying microbial strains, substrates, and fermentation processes are proposed toward a sustainable biological lignin valorization.

09 BIOMASS FUELS↗

DOE OSTI2020

Improved bacterial recombineering by parallelized protein discovery

Exploiting bacteriophage-derived homologous recombination processes has offered precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we present a high-throughput method for SSAP discovery that we call “serial enrichment for efficient recombineering” (SEER). By performing SEER in Escherichia coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in E. coli , while PapRecT enables efficient recombineering in Pseudomonas aeruginosa , a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis

Previously, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. In this paper, we developed a protoplast assay to rapidly optimize the multiplexed CRISPR/Cas9 system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples with 94% of deletions larger than 10bp, and less than 1% of mutations being insertions. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical NHEJ DNA repair pathway. Combined with the robust agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at the frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable MMEJ-mediated mutations in many plant species.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Turbo‐charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond

Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene‐free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High‐throughput sequencing technologies, including long‐read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements—often missed by standard genotyping—when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user‐friendly, synthetic biology‐compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue‐specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next‐generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies

Multiplexed CRISPR technologies have great potential for pathway engineering and genome editing. However, their applications are constrained by complex, laborious and time-consuming cloning steps. In this research, we developed a novel method, PARA, which allows for the one-step assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Here, we demonstrate that PARA is capable of the efficient assembly of transfer RNA/Csy4/ribozyme-based gRNA arrays. To aid in this process and to streamline vector construction, we developed a user-friendly PARAweb tool for designing PCR primers and component DNA parts and simulating assembled gRNA arrays and vector sequences.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Multiplex base editing to convert $\mathrm{TAG}$ into $\mathrm{TAA}$ codons in the human genome

Whole-genome recoding has been shown to enable nonstandard amino acids, biocontainment and viral resistance in bacteria. Here we take the first steps to extend this to human cells demonstrating exceptional base editing to convert TAG to TAA for 33 essential genes via a single transfection, and examine base-editing genome-wide (observing ~40 C-to-T off-target events in essential gene exons). We also introduce GRIT, a computational tool for recoding. This demonstrates the feasibility of recoding, and highly multiplex editing in mammalian cells.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

A landing pad system for multicopy gene integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. Furthermore, this study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

A Landing Pad System for Multicopy Gene Integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

Fatma, Zia↗

DOE OSTI2021

Construct design for CRISPR/Cas-based genome editing in plants

CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

In vivo human T cell engineering with enveloped delivery vehicles

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR–Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody–antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

42 ENGINEERING↗

DOE OSTI2023

Multiplex Editing of the Nucleoredoxin1 Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions

The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ~100 kb in Populus tremula × Populus alba. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

High-throughput functional variant screens via in vivo production of single-stranded DNA

Significance We report a methodology for the pooled construction of mutants bearing precise genomic sequence variations and multiplex phenotypic characterization of these mutants using next-generation sequencing (NGS). Unlike existing techniques depending on CRISPR-Cas–directed genomic breaks for genome editing, this strategy instead uses single-stranded DNA produced by a retron element for recombineering. This enables libraries of millions of elements to be constructed and offers relaxed design constraints which permit natural DNA or random variation to be used as inputs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Advances in genetic tools for metabolic engineering of non-conventional yeasts

Non-conventional yeasts are emerging as powerful alternatives to Saccharomyces cerevisiae for metabolic engineering, owing to their innate stress tolerance, broad substrate utilization, and distinctive metabolic capabilities. These attributes position them as promising chassis for producing biofuels, pharmaceuticals, and specialty chemicals. This review synthesizes recent advances in genetic toolkits for four such species—Pichia kudriavzevii (Issatchenkia orientalis), Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus—highlighting progress across plasmid architectures (episomal and integrative), identification of autonomously replicating sequences and centromeric elements, and the development of safe-harbor genomic loci. We summarize promoter and terminator libraries enabling tunable expression, the expansion of auxotrophic and antifungal selection markers with recycling strategies, and the rapid adaptation of CRISPR-based systems (Cas9 and Cas12a) with optimized guide RNA expression, multiplex editing, and approaches that enhance homologous recombination (e.g., KU70/80 disruption). We also review landing-pad platforms for modular, repeated integrations and transposon-based tools (e.g., piggyBac) that facilitate multigene pathway assembly. Collectively, these innovations are accelerating design-build-test-learn cycles and enabling precise, scalable engineering of non-conventional yeasts. Remaining challenges—including limited species-specific episomal systems, variable transformation efficiencies, genome-stability concerns, and alternative codon usage—define clear priorities for future toolkit development. Together, these advances and open needs chart a path toward robust, sustainable biomanufacturing using diverse non-conventional yeast chassis.

59 BASIC BIOLOGICAL SCIENCES↗