Genome Editing Methods Research Articles

Interspecific hybridization and cell engineering of lettuce (Lactuca L.)

Lactuca sativa L. is a leafy vegetable crop of the Asteraceae family, widely cultivated throughout the world. The main breeding trends for lettuce include higher yields, better taste quality, earliness, and resistance to abiotic and biotic stressors. Some wild Lactuca spp. have actively been employed by lettuce breeders as donors of resistance to various diseases. Conventional and biotechnological breeding methods are both currently used to develop new lettuce cultivars. This is an overview of the main advances in the production of interspecific Lactuca hybrids, including the use of cell and tissue culture techniques, and genetic engineering. Studying artificial hybridization and natural populations makes it possible to identify evolutionary relationships among various Lactuca spp. Somatic hybridization is an overlooked but promising technology in Lactuca breeding: it allows a breeder to obtain a wider range of variations, and is beyond the strict control by GMO laws. This technique faces problems associated with complicated protoplast regeneration and the loss of reproductive ability in hybrids. Genome-editing methods are more effective and better controllable, but society is still wary of any interference with the plant genome and legally regulates the sale of GM products as food. Thus, researchers are challenged with the task to improve these techniques.

CRISPR/Cas12a-based genome editing for cyanophage of Anabeana sp.

Efforts have been conducted on cyanobacterial genome editing, yet achieving genome editing in cyanophages remains challenging. Editing cyanophage genomes is crucial for understanding and manipulating their interactions with cyanobacterial hosts, offering potential solutions for controlling cyanobacterial blooms. In this study, we developed a streamlined CRISPR-Cas12a-based method for efficient cyanophage genome editing and then applied this method to the cyanophages A-1(L) and A-4(L) of Anabeana sp. PCC.7120. Multiple hypothetical genes were edited and knocked out from these two cyanophage genomes, generating viable mutants with varying capabilities to inhibit cyanobacterial growth. All these mutants displayed significant inhibitory effects on the host, indicating that these genes were non-essential for phage life cycle and the deletion led to little impairment of the cyanophages in infectious efficiency to their host. By iterative and simultaneous gene knockouts in cyanophage A-4(L), we achieved the minimal genome mutant with a 2400 bp reduction in genome size, representing a 5.75 % decrease compared to the wild type (WT). In conclusion, these cyanophage mutants can facilitate the identification of nonessential genes for cyanophages biology and the insertion of foreign genes for synthetic biology research. This advancement holds promise in addressing the widespread issue of water blooms and the associated environmental hazards.

Genome Editing Technologies towards Tomato Improvement: Recent Advances and Future Perspectives

Tomato (Solanum lycopersicon L.) is the world's second major vegetable crop and a superior model plant for studies on fruit biology. However, the changing climatic conditions are hugely impacting the yield and quality of tomato. CRISPR/Cas9 technology has been widely used in tomato breeding for enhanced disease resistance, herbicide tolerance, domestication and urban farming of wild tomato, and improved fruit yield and quality. Furthermore, new and advanced editing systems like Cas12a, Cas12b, base editing, and prime editing have been recently applied for high-precision tomato improvement. CRISPR variants, PAM-less genome editing, advanced transformation protocols, and gene delivery systems have played a critical role in fast breeding. This review offers an informative summary of recent progress in various genome editing methods and applications for improving tomatoes. It also focuses on critical issues, regulatory concerns, and prospects of genome editing platforms to improve tomato and allied crops.

Genome editing in angiosperm chloroplasts: targeted DNA double-strand break and base editing.

Chloroplasts are organelles that are derived from a photosynthetic bacterium and have their own genome. Genome editing is a recently developing technology that allows for specific modifications of target sequences. The first successful application of genome editing in chloroplasts was reported in 2021, and since then, this research field has been expanding. Although the chloroplast genome of several dicot species can be stably modified by a conventional method, which involves inserting foreign DNAs into the chloroplast genome via homologous recombination, genome editing offers several advantages over this method. In this review, we introduce genome editing methods targeting the chloroplast genome and describe their advantages and limitations. So far, CRISPR/Cas systems are inapplicable for editing the chloroplast genome because guide RNAs, unlike proteins, cannot be efficiently delivered into chloroplasts. Therefore, protein-based enzymes are used to edit the chloroplast genome. These enzymes contain a chloroplast-transit peptide, the DNA-binding domain of transcription activator-like effector nuclease (TALEN), or a catalytic domain that induces DNA modifications. To date, genome editing methods can cause DNA double-strand break or introduce C:G-to-T:A and A:T-to-G:C base edits at or near the target sequence. These methods are expected to contribute to basic research on the chloroplast genome in many species and to be fundamental methods of plant breeding utilizing the chloroplast genome.

Conquering Limitations: Exploring the Factors that Drive Successful Agrobacterium-Mediated Genetic Transformation of Recalcitrant Plant Species.

Agrobacterium-mediated transformation is a preferred method for genetic engineering and genome editing of plants due to its numerous advantages, although not all species exhibit transformability. Genetic engineering and plant genome editing methods are technically challenging in recalcitrant crop plants. Factors affecting the poor rate of transformation in such species include host genotype, Agrobacterium genotype, type of explant, physiological condition of the explant, vector, selectable marker, inoculation method, chemical additives, antioxidative compounds, transformation-enhancing compounds, medium formulation, optimization of culture conditions, and pre-treatments. This review provides novel insights into the key factors involved in gene transfer facilitated by Agrobacterium and proposes potential solutions to overcome existing barriers to transformation in recalcitrant species, thereby contributing to improvement programs for these species. This review introduces the key factors that impact the effectiveness of a molecular breeding program using Agrobacterium-mediated transformation, specifically focusing on recalcitrant plant species.

Improving Rice Quality by Regulating the Heading Dates of Rice Varieties without Yield Penalties.

The heading date, a critical trait influencing the rice yield and quality, has always been a hot topic in breeding research. Appropriately delaying the flowering time of excellent northern rice varieties is of great significance for improving yields and enhancing regional adaptability during the process for introducing varieties from north to south. In this study, genes influencing the heading date were identified through genome-wide association studies (GWAS). Using KenDao 12 (K12), an excellent cultivar from northern China, as the material, the specific flowering activator, OsMADS50, was edited using the genome-editing method to regulate the heading date to adapt to the southern planting environment. The results indicated that the osmads50 mutant line of K12 flowered about a week later, with a slight increase in the yield and good adaptability in the southern region in China. Additionally, the expressions of key flowering regulatory genes, such as Hd1, Ghd7, Ehd1, Hd3a, and RFT1, were reduced in the mutant plants, corroborating the delayed flowering phenotype. Yield trait analysis revealed that the primary factor for improved yield was an increase in the number of effective tillers, although there is potential for further enhancements in the seed-setting rate and grain plumpness. Furthermore, there were significant increases in the length-to-width ratio of the rice grains, fat content, and seed transparency, all contributing to an overall improvement in the rice quality. In summary, this study successfully obtained a rice variety with a delayed growth period through OsMADS50 gene editing, effectively implementing the strategy for adapting northern rice varieties to southern climates. This achievement significantly supports efforts to enhance the rice yield and quality as well as to optimize production management practices.

USING ARTIFICIAL INTELLIGENCE TO OPTIMIZE GENETIC RESEARCH

The purpose of the article is to analyse the main achievements and prospects for the introduction of artificial intelligence in genetics, to improve the efficiency of research and the reliability of the results obtained. The article analyses publications that reveal the areas of integration of neural networks into DNA fingerprinting, in particular PCR-PCR analysis, sequencing, FISH diagnostics, etc., increasing the sensitivity of these technologies and reducing the cost of research. The author also discusses technologies for processing huge data sets of Big Data and their effectiveness on the example of international projects in theoretical and applied genetics. Attention is paid to artificial intelligence in genomics, proteomics and genetic engineering, in particular, optimisation of GED (genome editing) methods based on CRISPR (short palindromic repeats regularly interspaced in groups) to achieve better accuracy of genome editing. Neural networks are important in creating guide RNAs (gRNAs) for CRISPR-Cas systems, which determine the direction of endonuclease function. In addition to specialised applications and resources, artificial intelligence generally improves research work by optimising many processes that used to take a lot of time and effort. Examples of neural network-based applications that are widely used by the scientific community are presented.

CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis.

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

Prospects for combining the methods of haploid biotechnology and genome editing to improve spiked grains of the <i>Triticeae</i> family (review)

Over the past few decades, haploid biotechnologies have become an integral part of breeding programs for many crops. Using the strategy of doubling haploids induced in the culture of gametic cells and tissues in vitro, through androgenesis, gynogenesis and distant hybridization, it became possible to significantly reduce the time for developing new varieties. Using the technology of doubled haploids, within one or two generations, it is possible to obtain aligned homozygous lines, which can both help speed up the breeding process and study several scientific and practical issues. Another promising tool for developing lines and samples with specified traits within several generations is genome editing (engineering) using various nuclease-based engineering complexes. The CRISPR/Cas9 genome editing technology, which came into use ten years ago, allows solving a wide variety of problems in plant functional genomics, including engineering resistance to biotic and abiotic stresses, improving productivity and product quality. The technology is better than the most known methods for improving varieties for the traits which have mono- or polygenic control, since it allows changing several genes simultaneously, which is important for polyploid species. An integral part of plant genome editing, as well as haploidogenesis technologies, is cell and tissue culture in vitro, which gives possibility for their combination. The combination of technologies allows producing homozygous plants with new gene-specific mutations, which improves genetic diversity and accelerates the selection of linear material with new economically valuable traits. The current review has summarized the experience of combining haploidy and genome editing methods in spiked grains of the Triticeae family. In addition to analyzing the current state, there have been considered the prospects for further development of technologies for obtaining haploids of wheat, barley, triticale, and rye with an edited genome.

CRISPR/Cas9-mediated genome editing technique to control fall armyworm (Spodoptera frugiperda) in crop plants with special reference to maize.

Fall Armyworm imposes a major risk to agricultural losses. Insecticides have historically been used to manage its infestations, but it eventually becomes resistant to them. To combat the pest, a more recent strategy based on the use of transgenic maize that expresses Bt proteins such as Cry1F from the bacteria has been used. Nonetheless, there have been numerous reports of Cry1F maize resistance in FAW populations. Nowadays, the more effective and less time-consuming genome editing method known as CRISPR/Cas9 technology has gradually supplanted these various breeding techniques. This method successfully edits the genomes of various insects, including Spodoptera frugiperda. On the other hand, this new technique can change an insect's DNA to overcome its tolerance to specific insecticides or to generate a gene drive. The production of plant cultivars resistant to fall armyworms holds great potential for the sustainable management of this pest, given the swift advancement of CRISPR/Cas9 technology and its varied uses. Thus, this review article discussed and critically assessed the use of CRISPR/Cas9 genome-editing technology in long-term fall armyworm pest management. However, this review study focuses primarily on the mechanism of the CRISPR-Cas9 system in both crop plants and insects for FAW management.

Obtaining cattle embryos using ready-made media of the scientific and production enterprise "Paneco"

The development and widespread practical application of in vitro embryo production technology will make it possible to replenish the herd population in a short time, preserve and replicate valuable genotypes, as well as create new genotypes using the method of genome editing. Livestock breeding is tasked with providing agriculture with a high–value herd in a fairly short time, while farmers are charged with replenishing the herd in a short time, which is possible only with the active introduction of assisted reproductive technologies, such as in vitro embryo production (IVP technology). The use of domestically produced cultural media in technology will ensure greater accessibility, reduced economic costs and the rapid development of breeding workin Russia. The purpose of the work was to evaluate the effectiveness of the IVP technology when using culture media from the domestic manufacturer SPE “PanEco” (Russia) to obtain cattle embryos in vitro. Our own protocol for obtaining cattle embryos was based on the protocols of culture media manufacturers SPE “PanEco” and IVF Bioscience (UK). Thus, for the first time using the line of media from the domestic manufacturer “PanEco” to obtain cattle embryos in vitro under incubation conditions at temperatures from plus 38.6 ◦C to plus 38.9 ◦C, CO2 content 6.2-6.8 % and uncontrolled humidity, on 6-8 days after a period of time from 140 to 182 hours from the moment of fertilization, 30.4 % of embryos were obtained at the blastocyst stage in relation to the total number of fertilized oocytes that reached the stage of 2 – 4 blastomeres.

Advancing crop disease resistance through genome editing: a promising approach for enhancing agricultural production.

Modern agriculture has encountered several challenges in achieving constant yield stability especially due to disease outbreaks and lack of long-term disease-resistant crop cultivars. In the past, disease outbreaks in economically important crops had a major impact on food security and the economy. On the other hand climate-driven emergence of new pathovars or changes in their host specificity further poses a serious threat to sustainable agriculture. At present, chemical-based control strategies are frequently used to control microbial pathogens and pests, but they have detrimental impact on the environment and also resulted in the development of resistant phyto-pathogens. As a replacement, cultivating engineered disease-resistant crops can help to minimize the negative impact of regular pesticides on agriculture and the environment. Although traditional breeding and genetic engineering have been instrumental in crop disease improvement but they have certain limitations such as labour intensity, time consumption, and low efficiency. In this regard, genome editing has emerged as one of the potential tools for improving disease resistance in crops by targeting multiple traits with more accuracy and efficiency. For instance, genome editing techniques, such as CRISPR/Cas9, CRISPR/Cas13, base editing, TALENs, ZFNs, and meganucleases, have proved successful in improving disease resistance in crops through targeted mutagenesis, gene knockouts, knockdowns, modifications, and activation of target genes. CRISPR/Cas9 is unique among these techniques because of its remarkable efficacy, low risk of off-target repercussions, and ease of use. Some primary targets for developing CRISPR-mediated disease-resistant crops are host-susceptibility genes (the S gene method), resistance genes (R genes) and pathogen genetic material that prevents their development, broad-spectrum disease resistance. The use of genome editing methods has the potential to notably ameliorate crop disease resistance and transform agricultural practices in the future. This review highlights the impact of phyto-pathogens on agricultural productivity. Next, we discussed the tools for improving disease resistance while focusing on genome editing. We provided an update on the accomplishments of genome editing, and its potential to improve crop disease resistance against bacterial, fungal and viral pathogens in different crop systems. Finally, we highlighted the future challenges of genome editing in different crop systems for enhancing disease resistance.

Enhanced eMAGE applied to identify genetic factors of nuclear hormone receptor dysfunction via combinatorial gene editing

Technologies that generate precise combinatorial genome modifications are well suited to dissect the polygenic basis of complex phenotypes and engineer synthetic genomes. Genome modifications with engineered nucleases can lead to undesirable repair outcomes through imprecise homology-directed repair, requiring non-cleavable gene editing strategies. Eukaryotic multiplex genome engineering (eMAGE) generates precise combinatorial genome modifications in Saccharomyces cerevisiae without generating DNA breaks or using engineered nucleases. Here, we systematically optimize eMAGE to achieve 90% editing frequency, reduce workflow time, and extend editing distance to 20 kb. We further engineer an inducible dominant negative mismatch repair system, allowing for high-efficiency editing via eMAGE while suppressing the elevated background mutation rate 17-fold resulting from mismatch repair inactivation. We apply these advances to construct a library of cancer-associated mutations in the ligand-binding domains of human estrogen receptor alpha and progesterone receptor to understand their impact on ligand-independent autoactivation. We validate that this yeast model captures autoactivation mutations characterized in human breast cancer models and further leads to the discovery of several previously uncharacterized autoactivating mutations. This work demonstrates the development and optimization of a cleavage-free method of genome editing well suited for applications requiring efficient multiplex editing with minimal background mutations.

CRISPR-based gene editing in plants: Focus on reagents and their delivery tools

Introduction: CRISPR-Cas9 technology has revolutionized plant genome editing, providing precise and efficient methods for genetic modification. This study focuses on the advancements and delivery of CRISPR-Cas9 in plant gene editing. Methods: A comprehensive search in scientific databases, including PubMed, ScienceDirect, and Google Scholar, was conducted to gather information on CRISPR-Cas9 gene editing and its delivery in precise gene modification in plants. Results: The evolving landscape of CRISPR nucleases has led to the development of innovative technologies, enhancing plant research. However, successful editing is contingent on efficient delivery of genome engineering reagents. CRISPR-based gene editing in plants utilizes diverse delivery methods: Agrobacterium-mediated transformation for bacterial transfer, biolistic transformation for physical gene insertion, electroporation for direct gene entry, expression of developmental regulators for gene expression modulation, and tobacco rattle virus as a viral vector, each offering distinct advantages for precise and efficient genetic modification in plants. Conclusion: CRISPR-Cas9 gene editing stands as a pivotal advancement in plant genetics, offering precise gene manipulation with applications in agriculture and biotechnology. The continuous refinement of reagent delivery tools reinforces CRISPR-Cas9's transformative role in plant genome editing, with significant implications for broader scientific applications.

In Search of a Target Gene for a Desirable Phenotype in Aquaculture: Genome Editing of Cyprinidae and Salmonidae Species.

Aquaculture supplies the world food market with a significant amount of valuable protein. Highly productive aquaculture fishes can be derived by utilizing genome-editing methods, and the main problem is to choose a target gene to obtain the desirable phenotype. This paper presents a review of the studies of genome editing for genes controlling body development, growth, pigmentation and sex determination in five key aquaculture Salmonidae and Cyprinidae species, such as rainbow trout (Onchorhynchus mykiss), Atlantic salmon (Salmo salar), common carp (Cyprinus carpio), goldfish (Carassius auratus), Gibel carp (Carassius gibelio) and the model fish zebrafish (Danio rerio). Among the genes studied, the most applicable for aquaculture are mstnba, pomc, and acvr2, the knockout of which leads to enhanced muscle growth; runx2b, mutants of which do not form bones in myoseptae; lepr, whose lack of function makes fish fast-growing; fads2, Δ6abc/5Mt, and Δ6bcMt, affecting the composition of fatty acids in fish meat; dnd mettl3, and wnt4a, mutants of which are sterile; and disease-susceptibility genes prmt7, gab3, gcJAM-A, and cxcr3.2. Schemes for obtaining common carp populations consisting of only large females are promising for use in aquaculture. The immobilized and uncolored zebrafish line is of interest for laboratory use.

Identification and Functional Analysis of KH Family Genes Associated with Salt Stress in Rice.

Salinity stress has a great impact on crop growth and productivity and is one of the major factors responsible for crop yield losses. The K-homologous (KH) family proteins play vital roles in regulating plant development and responding to abiotic stress in plants. However, the systematic characterization of the KH family in rice is still lacking. In this study, we performed genome-wide identification and functional analysis of KH family genes and identified a total of 31 KH genes in rice. According to the homologs of KH genes in Arabidopsis thaliana, we constructed a phylogenetic tree with 61 KH genes containing 31 KH genes in Oryza sativa and 30 KH genes in Arabidopsis thaliana and separated them into three major groups. In silico tissue expression analysis showed that the OsKH genes are constitutively expressed. The qRT-PCR results revealed that eight OsKH genes responded strongly to salt stresses, and OsKH12 exhibited the strongest decrease in expression level, which was selected for further study. We generated the Oskh12-knockout mutant via the CRISPR/Cas9 genome-editing method. Further stress treatment and biochemical assays confirmed that Oskh12 mutant was more salt-sensitive than Nip and the expression of several key salt-tolerant genes in Oskh12 was significantly reduced. Taken together, our results shed light on the understanding of the KH family and provide a theoretical basis for future abiotic stress studies in rice.

Transgenesis in microalga <i>Chlamydomonas reinhardtii</i>: current approaches

Microalgae are a rich source of biologically active substances of natural origin, which have potential for use in pharmaceutical, agricultural, food and industrial production. Genetic engineering of microalgae opens up great prospects for creating improved strains that produce various food additives, commercial enzymes, as well as proteins for therapeutic purposes — antibodies, hormones and vaccines. Chlamydomonas reinhardtii P.A. Dang. is a unicellular green alga, a reference organism for studying the genetics of photosynthesis and developing new genetic engineering approaches in microalgae. The advantages of C. reinhardtii include the ability to transform all three of its genomes (nuclear, mitochondrial and chloroplast), low cost and ease of cultivation, safety for humans and the presence of a system for post-translational modification of proteins, which makes this organism a potential platform for use in biotechnology. Over the past few years, significant advances have been made in transgenesis of C. reinhardtii, including the use of new techniques based on the CRISPR/Cas9 genome editing technology. In this review, we summarize the available information on current approaches to transgenesis of the unicellular green alga C. reinhardtii: 1) general principles of transgenic constructs design for transformation of the nuclear and chloroplast genome, 2) popular selection markers used, 3) methods of cell transformation, 4) methods of genome editing using the CRISPR/Cas9 system.

CRISPR-Cas9: An Evolutionary Technique for the Treatment of Breast and Cervical Carcinoma

CRISPR/Cas9 has transformed genome editing methods in several scientific domains, including the study of human cancer. Globally, breast cancer (BC) affects women more than any other kind of cancer and triple-negative breast cancer (TNBC) has the greatest fatality rate. The diagnosis and treatment of breast cancer have come a long way, yet many patients still experience metastases or relapses. One of the potential techniques for correcting faulty genes and curing different cancers is gene therapy. Certain mutations associated with cancer onset and development might be repaired in model organisms as a first step toward translational applications using the CRISPR/Cas9 technology. The CRISPR/Cas9 system, which consists of a single readily modified guide RNA (sgRNA) sequence coupled to a Cas9 nuclease, has transformed genome editing owing to its simplicity and effectiveness when compared to previous methods. CRISPR/Cas9 allows for the knockdown of over-expressed genes, the reversal of mutations in defected genes, and the remodeling of the regulatory environment to inhibit BC proliferation. CRISPR/Cas9-based treatment mixed with traditional chemotherapy has proven to be beneficial in combating drug resistance and tumor inhibition difficulties. The CRISPR cas9 system may edit many genes at the same time by generating numerous sgRNAs that target different genomic regions, considerably increasing its therapeutic potential and making it a unique technique for treating cervical lesions. This review focuses on the therapeutic applications of CRISPR/Cas9 in the treatment of cervical cancer and breast cancer.

Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock.

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.

An improved genome editing system for Sphingomonadaceae.

The sphingomonads encompass a diverse group of bacteria within the family Sphingomonadaceae, with the presence of sphingolipids on their cell surface instead of lipopolysaccharide as their main common feature. They are particularly interesting for bioremediation purposes due to their ability to degrade or metabolise a variety of recalcitrant organic pollutants. However, research and development on their full bioremediation potential has been hampered because of the limited number of tools available to investigate and modify their genome. Here, we present a markerless genome editing method for Sphingopyxis granuli TFA, which can be further optimised for other sphingomonads. This procedure is based on a double recombination triggered by a DNA double-strand break in the chromosome. The strength of this protocol lies in forcing the second recombination rather than favouring it by pressing a counterselection marker, thus avoiding laborious restreaking or passaging screenings. Additionally, we introduce a modification with respect to the original protocol to increase the efficiency of the screening after the first recombination event. We show this procedure step by step and compare our modified method with respect to the original one by deleting ecfG2, the master regulator of the general stress response in S. granuli TFA. This adds to the genetic tool repertoire that can be applied to sphingomonads and stands as an efficient option for fast genome editing of this bacterial group.