Genome Editing Research Articles

Genome editing in ubiquitous freshwater Actinobacteria.

To advance bioproduction or bioremediation in large, unsupervised environmental systems such as ponds, wastewater lagoons, or groundwater systems, it will be necessary to develop diverse genetically amenable microbial model organisms. Although we already genetically modify a few key species, tools for engineering more microbial taxa, with different natural phenotypes, will enable us to genetically engineer multispecies consortia or even complex communities. Developing genetic tools for modifying freshwater bacteria is particularly important, as wastewater, production ponds or raceways, and contaminated surface water are all freshwater systems where microbial communities are already deployed to do work, and the outputs could potentially be enhanced by genetic modifications. Here, we demonstrate that common tools for genome editing can be used to inactivate specific genes in two representatives of a very widespread, environmentally relevant group of Actinobacteria. These Actinobacteria are found in almost all tested surface freshwater environments, where they co-occur with primary producers, and genome-editing tools in these species are thus a step on the way to engineering microbial consortia in freshwater environments.

An Evolutionary Perspective on Dog Behavioral Genetics.

Dogs have played an outsized role in the field of behavioral genetics since its earliest days. Their unique evolutionary history and ubiquity in the modern world make them a potentially powerful model system for discovering how genetic changes lead to changes in behavior. Genomic technology has supercharged this potential by enabling scientists to sequence the DNA of thousands of dogs and test for correlations with behavioral traits. However, fractures in the early history of animal behavior between biological and psychological subfields may be impeding progress. In addition, canine behavioral genetics has included almost exclusively dogs from modern breeds, who represent just a small fraction of all dog diversity. By expanding the scope of dog behavior studies, and incorporating an evolutionary perspective on canine behavioral genetics, we can move beyond associations to understanding the complex interactions between genes and environment that lead to dog behavior.

Lipid nanoparticle-mediated base-editing of the Hao1 gene achieves sustainable primary hyperoxaluria type 1 therapy in rats.

Primary hyperoxaluria type 1 (PH1) is a severe hereditary disease, leading to the accumulation of oxalate in multiple organs, particularly the kidney. Hydroxyacid oxidase 1 (HAO1), a pivotal gene involved in oxalate production, is an approved target for the treatment of PH1. In this study, we demonstrated the discovery of several novel therapeutic sites of the Hao1 gene and the efficient editing of Hao1 c.290-2 A in vivo with lipid nanoparticles (LNP) delivered adenine base editing (ABE) mRNA. A single infusion of LNP-ABE resulted in a near-complete knockout of Hao1 in the liver, leading to the sustainable normalization of urinary oxalate (for at least 6 months) and complete rescue of the patho-physiology in PH1 rats. Additionally, a significant correlation between Hao1 editing efficiency and urinary oxalate levels was observed and over 60% Hao1 editing efficiency was required to achieve the normalization of urinary oxalate in PH1 rats. These findings suggest that the LNP-mediated base-editing of Hao1 c.290-2 A is an efficient and safe approach to PH1 therapy, highlighting its potential utility in clinical settings.

Modern alternative research methods in genetic toxicology (literature review)

The review represents the current principles of assessment of chemicals genotoxicity. The main attention is paid to alternative research methods. The international experience of the application of alternative approaches and prospects of their use for regulatory purposes are discussed. The data for this review were collected from the Russian and foreign literature, as well as Internet resources, concerning the development of the new alternative methods for testing chemicals for genotoxicity. The OECD database, Scopus, Medline, Google Scholar, RISC, CyberLeninka were used for the information retrieval. Although the evaluation of genotoxicity of chemical substances is the well-established and based on the battery of validated methods, the studies for improving the existing tests and developing new technologies, including the alternative approaches, continue unabated up to now. In general, three trends of development of genetic toxicology can be outlined, including creating of new methods based on the whole-genome sequencing and the application of genome editing technologies; implementation of quantitative system of effects assessment in addition to the existing qualitative approach (mutagenic/non-mutagenic) and testing of various combinations of genotoxicity evaluation methods to identify a battery of tests with a greater predictive activity regarding carcinogenic effects. To use the developed alternative models for regulatory purposes, it is necessary to provide convincing evidence that the data obtained are good predictors of the organism’s actual response to the effects of toxicants/genotoxicants, validation of methods, standardization, and harmonization of research protocols, and changes to the existing regulatory framework are required.

CRISPR-Cas systems and applications for crop bioengineering.

CRISPR-Cas technologies contribute to enhancing our understanding of plant gene functions, and to the precise breeding of crop traits. Here, we review the latest progress in plant genome editing, focusing on emerging CRISPR-Cas systems, DNA-free delivery methods, and advanced editing approaches. By illustrating CRISPR-Cas applications for improving crop performance and food quality, we highlight the potential of genome-edited crops to contribute to sustainable agriculture and food security.

SnPATHO-seq, a versatile FFPE single-nucleus RNA sequencing method to unlock pathology archives

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3’ and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization.

Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a type IV secretion system into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from DNA breaks produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The guide RNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild-type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides a delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells.

Be-dataHIVE: a base editing database

Base editing is an enhanced gene editing approach that enables the precise transformation of single nucleotides and has the potential to cure rare diseases. The design process of base editors is labour-intensive and outcomes are not easily predictable. For any clinical use, base editing has to be accurate and efficient. Thus, any bystander mutations have to be minimized. In recent years, computational models to predict base editing outcomes have been developed. However, the overall robustness and performance of those models is limited. One way to improve the performance is to train models on a diverse, feature-rich, and large dataset, which does not exist for the base editing field. Hence, we develop BE-dataHIVE, a mySQL database that covers over 460,000 gRNA target combinations. The current version of BE-dataHIVE consists of data from five studies and is enriched with melting temperatures and energy terms. Furthermore, multiple different data structures for machine learning were computed and are directly available. The database can be accessed via our website https://be-datahive.com/ or API and is therefore suitable for practitioners and machine learning researchers.

Genome Editing for Grass Improvement and Future Agriculture

Abstract Grasses, including turf and forage, cover most of the earth’s surface; predominantly important for land, water, livestock feed, soil and water conservation, as well as carbon sequestration. Improved production and quality of grasses by modern molecular breeding is gaining more research attention. Recent advances in genome-editing technologies are helping to revolutionize plant breeding and also offering smart and efficient acceleration on grass improvement. Here, we reviewed all recent researches using (CRISPR)/CRISPR-associated protein (Cas)-mediated genome editing tools to enhance the growth and quality of forage and turf grasses. Furthermore, we highlighted emerging approaches aimed at advancing grass breeding program. We assessed the CRISPR-Cas effectiveness, discussed the challenges associated with its application, and explored future perspectives primarily focusing on turf and forage grasses. Despite the promising potential of genome editing in grasses, its current efficiency remains limited due to several bottlenecks, such as the absence of comprehensive reference genomes, the lack of efficient gene delivery tools, unavailability of suitable vector and delivery for grass species, high polyploidization, multiple homoeoalleles, etc. Despite these challenges, the CRISPR-Cas system holds great potential to fully harness its benefits in grass breeding and genetics, aiming to improve and sustain the quantity and quality of turf and forage grasses.

Identification of lncRNAs regulating seed traits in Brassica juncea and development of a comprehensive seed omics database.

Brassica juncea is a crucial oilseed crop, and its seeds possess high economic value as they are a source of edible oil. In order to understand the role of long non coding RNAs (lncRNAs) in the regulation of seed development, we carried out computational analysis using transcriptome data of developing seeds of two contrasting genotypes of B. juncea, Pusajaikisan (PJK) and Early Heera 2 (EH2). The seeds were sampled at three stages, 15, 30, and 45 days after pollination. We identified 1,539 lncRNAs, of which 809 were differentially expressed. We also carried out extensive characterization and functional analysis of seed lncRNAome. The expression patterns were analysed using k-means clustering, and the targets were analysed using pathway, transcription factor, and GO enrichment, as well as ortholog information. We shortlisted a total of 25 robust lncRNA candidates for seed size, oil content, and seed coat color. We also identified 4 lncRNAs as putative precursors of miRNAs regulating seed development. Moreover, a total of 28 miRNA-lncRNA-mRNA regulatory networks regulating seed traits were identified. We also developed a comprehensive database, (BrassIca juncea database or "BIJ" ( https://bij.cuh.ac.in/ ), which provides seed omics as well as other functional genomics and genetics data in an easily accessible form. These candidate lncRNAs are suitable for including in crop improvement programs through molecular breeding, as well as for future validations through genome editing. Together, the knowledge of these candidate lncRNAs and availability of BIJ database shall leverage the crop improvement efforts in B. juncea.

Competition between homologous chromosomal DNA and exogenous donor DNA to repair CRISPR/Cas9-induced double-strand breaks in Aspergillus niger

BackgroundAspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.ResultsHere we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13–20% of the transformants while 80–87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35–61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85–90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.ConclusionThe awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

Establishment of a CIB1 knockout human pluripotent stem cell line via CRISPR/Cas9 genome editing technology

Calcium- and integrin-binding protein 1 (CIB1) has a diverse role in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. It is associated with cancer, cardiovascular disease and male infertility. Here, CRISPR/Cas9 genome-editing technology was employed to establish a CIB1 knockout human embryonic stem cell line, which exhibited normal pluripotency and karyotype.

A Review on Balancing Genomic Selection Efforts for Allogamous Plant Breeding

Genomic selection (GS) represents a paradigm shift in allogamous plant breeding, leveraging genome-wide marker data to predict the genetic potential of breeding candidates with unprecedented accuracy. This comprehensive review explores the evolution, principles, and applications of GS, highlighting its transformative impact on breeding efficiency and crop improvement. GS uses dense SNP markers to capture the genetic architecture of complex traits, surpassing traditional marker-assisted selection in predictive power. The integration of GS in breeding programs for crops like maize and perennial ryegrass has demonstrated significant gains in traits such as yield, disease resistance, and stress tolerance. Despite its advantages, GS faces challenges including high genotyping costs, the need for large training populations, and the complexity of genetic architectures. Economic constraints and ethical concerns about genetic diversity and data access also pose barriers. Emerging technologies such as AI, machine learning, high-throughput phenotyping, and genome editing hold promise for enhancing GS's accuracy and efficiency. Future strategies should focus on optimizing resource allocation, integrating GS with conventional breeding methods, and fostering collaborative efforts for data sharing and capacity building. The long-term impact of GS is profound, potentially accelerating breeding cycles, enhancing genetic diversity, and developing climate-resilient crops. Collaborative initiatives and open-access genomic resources will be crucial in overcoming current limitations and ensuring that GS benefits global agriculture. As GS continues to evolve, it promises to drive sustainable agricultural practices and improve food security, meeting the challenges posed by climate change and a growing global population. This review underscores the critical role of GS in modern plant breeding and its potential to revolutionize crop improvement.

CRISPR-GPT: An LLM Agent for Automated Design of Gene-Editing Experiments.

The introduction of genome engineering technology has transformed biomedical research, making it possible to make precise changes to genetic information. However, creating an efficient gene-editing system requires a deep understanding of CRISPR technology, and the complex experimental systems under investigation. While Large Language Models (LLMs) have shown promise in various tasks, they often lack specific knowledge and struggle to accurately solve biological design problems. In this work, we introduce CRISPR-GPT, an LLM agent augmented with domain knowledge and external tools to automate and enhance the design process of CRISPR-based gene-editing experiments. CRISPR-GPT leverages the reasoning ability of LLMs to facilitate the process of selecting CRISPR systems, designing guide RNAs, recommending cellular delivery methods, drafting protocols, and designing validation experiments to confirm editing outcomes. We showcase the potential of CRISPR-GPT for assisting non-expert researchers with gene-editing experiments from scratch and validate the agent's effectiveness in a real-world use case. Furthermore, we explore the ethical and regulatory considerations associated with automated gene-editing design, highlighting the need for responsible and transparent use of these tools. Our work aims to bridge the gap between biological researchers across various fields with CRISPR genome engineering technology and demonstrate the potential of LLM agents in facilitating complex biological discovery tasks.

Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy.

Gamma delta (γδ) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-MHC-associated antigens on transformed and pathogen-infected cells. In addition to their lack of alloreactivity, γδ T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of γδ T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene edited chimeric antigen receptor (CAR)-γδ T cells in support of therapeutic application. Engineered CAR-γδ T cells demonstrate high function in vitro and and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-γδ T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-γδ T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-γδ T cell subsets to therapeutic efficacy in vivo .

Using CRISPR-Cas9 genome editing to enhance disease resistance in banana

Abstract Bananas and plantains ( Musa spp.) are leading global fruit crops, cultivated in over 150 countries and several island nations and serving as fruit and staple food in tropical and subtropical regions. Notwithstanding their economic importance, banana production is hindered by pests, diseases, and climatic challenges, resulting in significant yield gaps and post-harvest losses. Conventional breeding methods are time-consuming and labor-intensive due to the 15–18 months long life cycle and seedless nature of the edible crop, while transgenic approaches face regulatory, public acceptance, and sociopolitical hurdles. The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) technology offers a precise, targeted, and efficient tool for genetic mutation derived from the bacterial antiviral defense system. Researchers are exploring CRISPR-Cas9 to develop banana varieties resistant to diseases and pests, exhibit higher yields, and possess enhanced nutritional profiles. The well-annotated complete banana genome, combined with advanced regeneration and transformation systems, facilitates the application of CRISPR-Cas9 technology. This innovative approach promises to create superior banana cultivars that benefit consumers and farmers, addressing critical challenges in banana production and contributing to global food security. This review article discusses the challenges in banana production, including biotic and abiotic constraints, and explores recent advancements in genome editing.

Effect of a loss of the mda5/ifih1 gene on the antiviral resistance in a Chinook salmon Oncorhynchus tshawytscha cell line.

Cells are equipped with intracellular RIG-like Receptors (RLRs) detecting double stranded (ds)RNA, a molecule with Pathogen-Associated Molecular Pattern (PAMPs) generated during the life cycle of many viruses. Melanoma Differentiation-Associated protein 5 (MDA5), a helicase enzyme member of the RLRs encoded by the ifih1 gene, binds to long dsRNA molecules during a viral infection and initiates production of type I interferon (IFN1) which orchestrates the antiviral response. In order to understand the contribution of MDA5 to viral resistance in fish cells, we have isolated a clonal Chinook salmon Oncorhynchus tshawytscha epithelial-like cell line invalidated for the ifih1 gene by CRISPR/Cas9 genome editing. We demonstrated that IFN1 induction is impaired in this cell line after infection with the Snakehead Rhabdovirus (SHRV), the Salmon Alphavirus (SAV) or Nervous Necrosis Virus (NNV). The cell line, however, did not show any increase in cytopathic effect when infected with SHRV or SAV. Similarly, no cytopathic effect was observed in the ifih1-/- cell line when infected with Infectious Pancreatic Necrosis Virus (IPNV), Infectious Haemorrhagic Necrotic Virus (IHNV). These results indicate the redundancy of the antiviral innate defence system in CHSE-derived cells, which helps with circumventing viral evasion strategies.

Chromatin interaction maps of human arterioles reveal new mechanisms for the genetic regulation of blood pressure.

Arterioles are small blood vessels located just upstream of capillaries in nearly all tissues. The constriction and dilation of arterioles regulate tissue perfusion and are primary determinants of systemic blood pressure (BP). Abnormalities in arterioles are central to the development of major diseases such as hypertension, stroke, and microvascular complications of diabetes. Despite the broad and essential role of arterioles in physiology and disease, current knowledge of the functional genomics of arterioles is largely absent, partly because it is challenging to obtain and analyze human arteriole samples. Here, we report extensive maps of chromatin interactions, single-cell expression, and other molecular features in human arterioles and uncover new mechanisms linking human genetic variants to gene expression in vascular cells and the development of hypertension. Compared to large arteries, arterioles exhibited a higher proportion of pericytes which were strongly associated with BP traits. BP-associated single nucleotide polymorphisms (SNPs) were enriched in chromatin interaction regions in arterioles, particularly through enhancer SNP-promoter interactions, which were further linked to gene expression specificity across tissue components and cell types. Using genomic editing in animal models and human induced pluripotent stem cells, we discovered novel mechanisms linking BP-associated noncoding SNP rs1882961 to gene expression through long-range chromatin contacts and revealed remarkable effects of a 4-bp noncoding genomic segment on hypertension in vivo. We anticipate that our rich data and findings will advance the study of the numerous diseases involving arterioles. Moreover, our approach of integrating chromatin interaction mapping in trait-relevant tissues with SNP analysis and in vivo and in vitro genome editing can be applied broadly to bridge the critical gap between genetic discoveries and physiological understanding.

A comprehensive review of livestock development: insights into domestication, phylogenetics, diversity, and genomic advances.

Livestock plays an essential role in sustaining human livelihoods, offering a diverse range of species integral to food security, economic stability, and cultural traditions. The domestication of livestock, which began over 10,000years ago, has driven significant genetic changes in species such as cattle, buffaloes, sheep, goats, and pigs. Recent advancements in genomic technologies, including next-generation sequencing (NGS), genome-wide association studies (GWAS), and genomic selection, have dramatically enhanced our understanding of these genetic developments. This review brings together key research on the domestication process, phylogenetics, genetic diversity, and selection signatures within major livestock species. It emphasizes the importance of admixture studies and evolutionary forces like natural selection, genetic drift, and gene flow in shaping livestock populations. Additionally, the integration of machine learning with genomic data offers new perspectives on the functional roles of genes in adaptation and evolution. By exploring these genomic advancements, this review provides insights into genetic variation and evolutionary processes that could inform future approaches to improving livestock management and adaptation to environmental challenges, including climate change.

An efficient CRISPR-Cas12a-mediated MicroRNA knockout strategy in plants.

In recent years, the CRISPR-Cas9 nuclease has been used to knock out MicroRNA (miRNA) genes in plants, greatly promoting the study of miRNA function. However, due to its propensity for generating small insertions and deletions, Cas9 is not well-suited for achieving a complete knockout of miRNA genes. By contrast, CRISPR-Cas12a nuclease generates larger deletions, which could significantly disrupt the secondary structure of pre-miRNA and prevent the production of mature miRNAs. Through the case study of OsMIR390 in rice, we confirmed that Cas12a is a more efficient tool than Cas9 in generating knockout mutants of a miRNA gene. To further demonstrate CRISPR-Cas12a-mediated knockout of miRNA genes in rice, we targeted nine OsMIRNA genes that have different spaciotemporal expression and have not been previously investigated via genetic knockout approaches. With CRISPR-Cas12a, up to 100% genome editing efficiency was observed at these miRNA loci. The resulting larger deletions suggest Cas12a robustly generated null alleles of miRNA genes. Transcriptome profiling of the miRNA mutants, as well as phenotypic analysis of the rice grains revealed the function of these miRNAs in controlling gene expression and regulating grain quality and seed development. This study established CRISPR-Cas12a as an efficient tool for genetic knockout of miRNA genes in plants.