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All sources NASA NTRS DOE OSTI

DOE OSTI2022

Cas9-induced large deletions and small indels are controlled in a convergent fashion

Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Benchmarking Computational Tools for Calling SNPs and Indels in Complex Microbial Populations

The NASA BioNutrients missions seek to understand the suitability of microorganisms for bioproduction during space flight. One topic of interest is the stability of microbial genomes during long-term ambient storage and subsequent rehydration and growth. To address these questions, samples from 8 species were flown to ISS for 5 years of desiccated storage at ambient temperature (Stasis Packs) and 2 species were packaged along with powdered media inside a bioreactor system to allow hydration and growth in microgravity (Production Packs). For both systems, Whole Genome Sequencing (WGS) of the DNA extracted from the returned samples and paired ground controls will be conducted to identify changes in genome stability due to time, storage conditions and growth in space. Across the technical replicates, ground controls, 10 timepoints, and multiple experimental conditions, ~300 samples have been selected for initial analysis with WGS sequencing to 100x coverage. A flexible and resource efficient mutation calling pipeline is needed to process this large dataset and allow for comparisons between species. Many bioinformatics tools for calling Indels and Single Nucleotide Variants (SNVs) are designed for use with pure isolates, where true variations from the reference genome are expected to dominate the reads aligning to the location of mutation. In contrast, DNA from the Stasis Pack (SP) samples was collected directly after recovery from desiccated storage and the Production Pack (PP) samples were collected after fermentation. In this context, reads with mutations are expected to be less frequent than reads that align with the reference genome, as each sample will include multiple lines of cells. Thus, BioNutrients samples are expected to be similar to samples from cancer cell or “pooled” sequencing approaches. In preparation for the analysis of the BioNutrients samples, we have tested three mutation calling tools (GATK for Microbes, BreSeq and DiscoSNP) designed for complex samples. A challenge of validating mutation identification pipelines is a lack of “Ground Truth” datasets, especially for complex samples. To compare these three tools, we sought to identify mutations in pre-existing WGS data collected from populations of Chlamydomonas reinhardtii that were exposed to UV mutagenesis and growth in LEO as part of the Space Algae-1 mission. Here we present a summary of these tools against the analysis originally conducted using the CRISP tool. Critical metrics are compared such as runtime, the number of SNPs, the number and size of Indels, and patterns of transversion and transitions identified by each tool are reported. By sharing these benchmarking results collected in support of the BioNutrients mission, we aim to guide others seeking to identify SNVs in similarly complex microbial samples.

Biology↗

DOE OSTI2024

BAD2matrix: Phylogenomic matrix concatenation, indel coding, and more

Common steps in phylogenomic matrix production include biological sequence concatenation, morphological data concatenation, insertion/deletion (indel) coding, gene content (presence/absence) coding, removing uninformative characters for parsimony analysis, recording with reduced amino acid alphabets, and occupancy filtering. Existing software does not accomplish these tasks on a phylogenomic scale using a single program. BAD2matrix is a Python script that performs the above-mentioned steps in phylogenomic matrix construction for DNA or amino acid sequences as well as morphological data. The script works in UNIX-like environments (e.g., LINUX, MacOS, Windows Subsystem for LINUX).

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

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 OSTI2025

Supplementary table, figures and DNA sequences of sorghum gene models SbiRTx430.01G455400 and SbiRTx.02G006600 that feature primers, gRNAs and indels created

In-context promoter bashing via genome editing is a route to identify and characterize critical regulatory regions that govern expression of genes of interest. The outcomes of in-context promoter bashing can be used to inform editing strategies to modulate the expression of selected gene models in a desired fashion. Here we employed in-context promoter bashing to characterize the proximal upstream regulatory regions of sorghum genes encoding phosphoenolpyruvate carboxykinase (Sb.PEPCK.BS, SbiTx430.01G455400) and alanine aminotransferase (SbiTx430.02G006600, SbAlaAT.BS), two proteins involved in the PCK C4 pathway. Characterized germinal edits within the targeted regions upstream of these two genes ranged in size from 138 bp up to 1790 bp. A 138 bp within the Sb.PEPCK.BS upstream region and a 1643 bp element within the Sb.AlaAT.BS upstream region were determined to be important for maintenance of transcription levels. No change in development or various physiological parameters was observed in characterized lineages carrying promoter edits. However, significant changes in seed reserves and a reduction in 100 seed weight were consistently observed, under both greenhouse and field environments, in plants carrying an edit in the promoter of Sb.PEPCK.BS gene were significantly reduced in transcript accumulation for this gene.

Quach, Truyen [Center for Plant Science Innovation↗

DOE OSTI2025

Energy metric prediction for double insertion mutants via the RoseNet deep learning framework

Studying the structural and functional implications of protein mutations is an important task in computational biology and bioinformatics. We leverage our previously proposed RoseNet neural network architecture to predict energy metrics of proteins with double amino acid insertions or deletions (InDels). We train models on previously generated benchmark datasets containing the exhaustive double InDel mutations for three proteins, as well as an additional three proteins for which ∼145k random mutants, each with two InDels, have been generated. We expand on our previous work by evaluating three additional proteins and analyzing domain features that impact the prediction capabilities of RoseNet. These features include InDels into secondary structures and the solvent accessible surface area (SASA) scores of the residues. We uncover further evidence to support that RoseNet has a higher proficiency of generalizing to unseen residue combinations than unseen insertion positions. We also observe that RoseNet produces higher-quality predictions when inserting into a β-sheet over an α-helix. Additionally, when the insertions fall in an area of high SASA, RoseNet often displays better performance than inserting into areas of low SASA.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Decorating chromatin for enhanced genome editing using CRISPR-Cas9

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Spectra and characteristics of somatic mutations induced by ionizing radiation in hematopoietic stem cells

Spectra and frequencies of spontaneous and X-ray-induced somatic mutations were revealed with mouse long-term hematopoietic stem cells (LT-HSCs) by whole-genome sequencing of clonal cell populations propagated in vitro from single isolated LT-HSCs. SNVs and small indels were the most common types of somatic mutations, and increased up to twofold to threefold by whole-body X-irradiation. Base substitution patterns in the SNVs suggested a role of reactive oxygen species in radiation mutagenesis, and signature analysis of single base substitutions (SBS) revealed a dose-dependent increase of SBS40. Most of spontaneous small deletions were shrinkage of tandem repeats, and X-irradiation specifically induced small deletions out of tandem repeats (non-repeat deletions). Presence of microhomology sequences in non-repeat deletions suggested involvement of microhomology mediated end-joining repair mechanisms as well as nonhomologous end-joining in radiation-induced DNA damages. We also identified multisite mutations and structural variants (SV), i.e., large indels, inversions, reciprocal translocations, and complex variants. The radiation-specificity of each mutation type was evaluated from the spontaneous mutation rate and the per-Gy mutation rate estimated by linear regression, and was highest with non-repeat deletions without microhomology, followed by those with microhomology, SV except retroelement insertions, and multisite mutations; these types were thus revealed as mutational signatures of ionizing radiation. Further analysis of somatic mutations in multiple LT-HSCs indicated that large fractions of postirradiation LT-HSCs originated from single LT-HSCs that survived the irradiation and then expanded in vivo to confer marked clonality to the entire hematopoietic system, with varying clonal expansion and dynamics depending on radiation dose and fractionation.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗

DOE OSTI2024

Sensitive and error-tolerant annotation of protein-coding DNA with BATH

We present BATH, a tool for highly sensitive annotation of protein-coding DNA based on direct alignment of that DNA to a database of protein sequences or profile hidden Markov models (pHMMs). BATH is built on top of the HMMER3 code base, and simplifies the annotation workflow for pHMM-based translated sequence annotation by providing a straightforward input interface and easy-to-interpret output. BATH also introduces novel frameshift-aware algorithms to detect frameshift-inducing nucleotide insertions and deletions (indels). BATH matches the accuracy of HMMER3 for annotation of sequences containing no errors, and produces superior accuracy to all tested tools for annotation of sequences containing nucleotide indels. These results suggest that BATH should be used when high annotation sensitivity is required, particularly when frameshift errors are expected to interrupt protein-coding regions, as is true with long-read sequencing data and in the context of pseudogenes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Vanderwaltozyma urihicola sp. nov., a yeast species isolated from rotting wood and beetles in a Brazilian Amazonian rainforest biome

Five yeast isolates belonging to a candidate for novel species were obtained from rotting wood and the gut of a passalid beetle larva in a site of Amazonian rainforest biome in Brazil. Sequence analysis of the Internal Transcribed Spacer (ITS)-5.8S region and the D1/D2 domains of the large subunit rRNA gene showed that the isolates represent a novel species of the genus Vanderwaltozyma. The closest relative of the novel species is Vanderwaltozyma huisunica. These species differs due to 44 nt substitutions and 21 indels in the sequences of the ITS region, as well as by 15 substitutions and four indels in the sequences of the D1/D2 domains. A phylogenomic analysis of the Vanderwaltozyma species with genomes sequenced showed that this novel species is an outgroup to the other species of this genus. We propose the name Vanderwaltozyma urihicola sp. nov. (CBS 18107T, MycoBank MB 856975) to accommodate these isolates. Furthermore, the species is homothallic, producing one to two ascospores per ascus. The habitat of V. urihicola is rotting wood in the Brazilian Amazonian rainforest biome.

Amazonian Forest↗

DOE OSTI2022

Sorghum Association Panel whole‐genome sequencing establishes cornerstone resource for dissecting genomic diversity

SUMMARY Association mapping panels represent foundational resources for understanding the genetic basis of phenotypic diversity and serve to advance plant breeding by exploring genetic variation across diverse accessions. We report the whole‐genome sequencing (WGS) of 400 sorghum ( Sorghum bicolor (L.) Moench) accessions from the Sorghum Association Panel (SAP) at an average coverage of 38× (25–72×), enabling the development of a high‐density genomic marker set of 43 983 694 variants including single‐nucleotide polymorphisms (approximately 38 million), insertions/deletions (indels) (approximately 5 million), and copy number variants (CNVs) (approximately 170 000). We observe slightly more deletions among indels and a much higher prevalence of deletions among CNVs compared to insertions. This new marker set enabled the identification of several novel putative genomic associations for plant height and tannin content, which were not identified when using previous lower‐density marker sets. WGS identified and scored variants in 5‐kb bins where available genotyping‐by‐sequencing (GBS) data captured no variants, with half of all bins in the genome falling into this category. The predictive ability of genomic best unbiased linear predictor (GBLUP) models was increased by an average of 30% by using WGS markers rather than GBS markers. We identified 18 selection peaks across subpopulations that formed due to evolutionary divergence during domestication, and we found six F st peaks resulting from comparisons between converted lines and breeding lines within the SAP that were distinct from the peaks associated with historic selection. This population has served and continues to serve as a significant public resource for sorghum research and demonstrates the value of improving upon existing genomic resources.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Identification of a Fusarium ear rot resistance gene in maize by QTL mapping and RNA sequencing

Fusarium ear rot (FER) caused by Fusarium verticillioides is a prevalent maize disease. To comprehensively characterize the genetic basis of the natural variation in FER resistance, a recombinant inbred line (RIL) population was used to map quantitative trait loci (QTL) for FER resistance. A total of 17 QTL were identified by linkage mapping in eight environments. These QTL were located on six chromosomes and explained 3.88–15.62% of the total phenotypic variation. Moreover, qFER1.03 had the strongest effect and accounted for 4.98–15.62% of the phenotypic variation according to analyses of multiple environments involving best linear unbiased predictions. The chromosome segment substitution lines (CSSLs) derived from a cross between Qi319 (donor parent) and Ye478 (recurrent parent) were used to verify the contribution of qFER1.03 to FER resistance. The line CL171, which harbored an introgressed qFER1.03 , was significantly resistant to FER. Further fine mapping of qFER1.03 revealed that the resistance QTL was linked to insertion/deletion markers InDel 8 and InDel 2, with physical distances of 43.55 Mb and 43.76 Mb, respectively. Additionally, qFER1.03 differed from the previous resistance QTL on chromosome 1. There were three annotated genes in this region. On the basis of the RNA-seq data, which revealed the genes differentially expressed between the FER-resistant Qi319 and susceptible Ye478, GRMZM2G017792 (MPK3) was preliminarily identified as a candidate gene in the qFER1.03 region. The Pr-CMV-VIGS system was used to decrease the GRMZM2G017792 expression level in CL171 by 34–57%, which led to a significant decrease in FER resistance. Using RIL and CSSL populations combined with RNA-seq and Pr-CMV-VIGS, the candidate gene can be dissected effectively, which provided important gene resource for breeding FER-resistant varieties.

Xia, Yusheng↗

DOE OSTI2020

Prime Editing Technology and Its Prospects for Future Applications in Plant Biology Research

Many applications in plant biology requires editing genomes accurately including correcting point mutations, incorporation of single-nucleotide polymorphisms (SNPs), and introduction of multinucleotide insertion/deletions (indels) into a predetermined position in the genome. These types of modifications are possible using existing genome-editing technologies such as the CRISPR-Cas systems, which require induction of double-stranded breaks in the target DNA site and the supply of a donor DNA molecule that contains the desired edit sequence. However, low frequency of homologous recombination in plants and difficulty of delivering the donor DNA molecules make this process extremely inefficient. Another kind of technology known as base editing can perform precise editing; however, only certain types of modifications can be obtained, e.g., C/G-to-T/A and A/T-to-G/C. Recently, a new type of genome-editing technology, referred to as “prime editing,” has been developed, which can achieve various types of editing such as any base-to-base conversion, including both transitions (C→T, G→A, A→G, and T→C) and transversion mutations (C→A, C→G, G→C, G→T, A→C, A→T, T→A, and T→G), as well as small indels without the requirement for inducing double-stranded break in the DNA. Because prime editing has wide flexibility to achieve different types of edits in the genome, it holds a great potential for developing superior crops for various purposes, such as increasing yield, providing resistance to various abiotic and biotic stresses, and improving quality of plant product. In this review, we describe the prime editing technology and discuss its limitations and potential applications in plant biology research.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2003

Implied alignment: a synapomorphy-based multiple-sequence alignment method and its use in cladogram search

A method to align sequence data based on parsimonious synapomorphy schemes generated by direct optimization (DO; earlier termed optimization alignment) is proposed. DO directly diagnoses sequence data on cladograms without an intervening multiple-alignment step, thereby creating topology-specific, dynamic homology statements. Hence, no multiple-alignment is required to generate cladograms. Unlike general and globally optimal multiple-alignment procedures, the method described here, implied alignment (IA), takes these dynamic homologies and traces them back through a single cladogram, linking the unaligned sequence positions in the terminal taxa via DO transformation series. These "lines of correspondence" link ancestor-descendent states and, when displayed as linearly arrayed columns without hypothetical ancestors, are largely indistinguishable from standard multiple alignment. Since this method is based on synapomorphy, the treatment of certain classes of insertion-deletion (indel) events may be different from that of other alignment procedures. As with all alignment methods, results are dependent on parameter assumptions such as indel cost and transversion:transition ratios. Such an IA could be used as a basis for phylogenetic search, but this would be questionable since the homologies derived from the implied alignment depend on its natal cladogram and any variance, between DO and IA + Search, due to heuristic approach. The utility of this procedure in heuristic cladogram searches using DO and the improvement of heuristic cladogram cost calculations are discussed. c2003 The Willi Hennig Society. Published by Elsevier Science (USA). All rights reserved.

Non-NASA Center↗

DOE OSTI2022

The Arabidopsis T‐DNA mutant SALK_008491 carries a 14‐kb deletion on chromosome 3 that provides rare insights into the plant response to dynamic light stress

Abstract In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light‐dependent reaction and CO 2 fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer‐DNA (T‐DNA) insertion mutant line SALK_008491, previously known as nhd1‐1 . Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non‐photochemical quenching (NPQ). Interestingly, an independent nhd1 null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F 2 pool, we identified an ~14‐kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of NHD1 . Besides NHD1 , which encodes for a putative plastid Na + /H + antiporter, the stromal NAD‐dependent D‐3‐phosphoglycerate dehydrogenase 3 ( PGDH3 ) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow‐up studies employing respective single mutants and complementation with overlapping transformation‐competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion‐carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T‐DNA lines. Although second‐site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T‐DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.

Lopez, Laura S.↗

DOE OSTI2023

Proteogenomic insights suggest druggable pathways in endometrial carcinoma

We characterized a prospective endometrial carcinoma (EC) cohort containing 138 tumors and 20 enriched normal tissues using 10 different omics platforms. Targeted quantitation of two peptides can predict antigen processing and presentation machinery activity, and may inform patient selection for immunotherapy. Association analysis between MYC activity and metformin treatment in both patients and cell lines suggests a potential role for metformin treatment in non-diabetic patients with elevated MYC activity. PIK3R1 in-frame indels are associated with elevated AKT phosphorylation and increased sensitivity to AKT inhibitors. CTNNB1 hotspot mutations are concentrated near phosphorylation sites mediating pS45-induced degradation of β-catenin, which may render Wnt-FZD antagonists ineffective. Deep learning accurately predicts EC subtypes and mutations from histopathology images, which may be useful for rapid diagnosis. Overall, this study identified molecular and imaging markers that can be further investigated to guide patient stratification for more precise treatment of EC.

60 APPLIED LIFE SCIENCES↗