Gene Editing System Research Articles

CRISPR-Cas9 Mediated Gene Editing Platform Through Callus-to-Plant Regeneration and Functional Analysis of DoALA4─DoALA6 in Dendrobium officinale.

Dendrobium orchids are well known for their great horticultural and medicinal values; however, the CRISPR/Cas9 gene editing system for Dendrobium species still needs to be improved. Therefore, this study aims to establish a CRISPR/Cas9-based functional validation system using Dendrobium officinale as a model species for the Dendrobium genus and to validate the DoALA4─DoALA6 genes, which may relate to growth and disease resistance. We first conducted a bioinformatics analysis of the P-type ATPase gene family in D. officinale, revealing the evolutionary diversity of P-type ATPase genes in orchids. Second, we inserted the GFP gene into the vector of CRISPR/Cas9 gene editing system to enhance the selection efficiency of genome-edited plants. Comparative analysis showed that different explants exhibited varying transformation efficiencies, ranging from 5% to 46.2%. Considering the regeneration capability, survival rate and gene editing efficiency, we selected callus as the transformation explant. Third, we used this editing system to generate DoALA4─DoALA6 mutants. Phenotypic observations of the mutants and inoculation of D. officinale with Sclerotium rolfsii indicated that DoALA4─DoALA6 are crucial for the growth of D. officinale and its resistance to southern blight disease. This efficient and stable CRISPR/Cas9 platform offers a foundation for further gene studies and Dendrobium breeding.

Probiotics Bi-Enzymatic Cascade Repair System for Editing the Inflammatory Microenvironment to Boost Probiotic Therapy in Inflammatory Bowel Disease.

Inflammatory bowel disease presents significant treatment challenges owing to its complex pathology. Although probiotics have shown promise as a therapeutic option, their effectiveness is often limited by low concentrations at sites of inflammation, exacerbated by excessive reactive oxygen species and inflammatory triggers. To address this, an innovative cascade repair system is developed to enhance probiotic therapeutic impact by modulating the intestinal microenvironment. This system uses iMXene's catalytic properties to neutralize reactive oxygen species in the gut and its capacity to deliver the CRISPR/dCas9 gene editing system to activate the NLR family pyrin domain containing 12 genes, helping suppress inflammation. By promoting the colonization of Lactobacillus rhamnosus, the system inhibits inflammation pathways and supports the restoration of a balanced intestinal flora through a cascade repair mechanism. These findings demonstrate significant therapeutic benefits in experimental models, with improvements in the overall well-being of treated mice and effective repair of intestinal inflammation damage. This pioneering approach holds promise for inflammatory bowel disease treatment and opens new avenues for managing other inflammatory conditions, offering valuable insights and guidance for future research into inflammatory diseases.

CRISPR/Cas9-Mediated BocPDSs Gene Editing in Chinese Kale Using the Endogenous tRNA-Processing System

Chinese kale is a native vegetable from the Brassicaceae family that is grown extensively in Southeast Asia and Southern China. Its low genetic transformation and gene editing efficiency hinder gene function research and molecular biology in Chinese kale. CRISPR/Cas9 is a useful tool for plant genome research due to its rapid development and optimization. This study targeted BocPDSs, (BocPDS1, BocPDS2) to establish an effective CRISPR/Cas9 system in Chinese kale. A tandemly arranged tRNA-sgRNA construct was used to express numerous sgRNAs to induce BocPDS1 and BocPDS2 double and single mutations, with a mutation rate of 61.11%. As predicted, several mutant plants showed an albino phenotype with a harbored mutation in an exon and intron region, highlighting the relevance of the intron. The presence of mutations in the intron region suggests that the cleavage process in Chinese kale, utilizing CRISPR/Cas9 shows a preference for AT-rich regions. The distinct and somewhat redundant functions of BocPDS1 and BocPDS2 are demonstrated by the complete albino phenotype of the double mutants and the mosaic albino phenotype of the individual BocPDS1 and BocPDS2 mutants. Specific gene editing modes, including base deletion, base substitution, and base insertion, were identified in the sequence of the target gene. Among them, short nucleotide insertions were the most common type of insertion, with base insertions having the highest frequency (61.54%). Furthermore, no instances of off-target gene editing were detected. The current work demonstrated that the CRISPR/Cas9 gene editing system, which relies on endogenous tRNA processing, can effectively induce mutagenesis in Chinese kale. This finding establishes a theoretical basis and technical backbone for the more effective implementation of CRISPR/Cas9 gene-editing technology in Chinese kale and Brassica plants.

Instrumentation and application of the CRISPR-Cas system

Abstract. The CRISPR-Cas gene editing system has revolutionized molecular biology by providing a versatile and precise method for genetic manipulation. We explore the basic mechanisms of CRISPR-Cas technology, focusing on the role of Cas proteins and guide RNAs in targeting and cleaving specific DNA sequences. Various types of CRISPR-Cas systems are also discussed, focusing on the widely used CRISPR-Cas9, and their applications in genome editing, including gene knockout, gene knock-in, and gene regulation. Additionally, we present advances in CRISPR-Cas technologies, such as base editing and plasmid editing, demonstrating their potential for therapeutic applications. This paper aims to provide a comprehensive overview of the status and applications of CRISPR-Cas technology and its future directions in gene editing.

Targeted deletion of olfactory receptors in D. melanogaster via CRISPR/Cas9-mediated LexA knock-in

The study of olfaction in Drosophila melanogaster has greatly benefited from genetic reagents such as olfactory receptor mutant lines and GAL4 reporter lines. The CRISPR/Cas9 gene-editing system has been increasingly used to create null receptor mutants or replace coding regions with GAL4 reporters. To further expand this toolkit for manipulating fly olfactory receptor neurons (ORNs), we generated null alleles for 11 different olfactory receptors by using CRISPR/Cas9 to knock in LexA drivers, including multiple lines for receptors which have thus far lacked knock-in mutants. The targeted neuronal types represent a broad range of antennal ORNs from all four morphological sensillum classes. Additionally, we confirmed their loss-of-function phenotypes, assessed receptor haploinsufficiency, and evaluated the specificity of the LexA knock-in drivers. These receptor mutant lines have been deposited at the Bloomington Drosophila Stock Center for use by the broader scientific community.

Tomato MADS-RIN regulates GAME5 expression to promote non-bitter glycoalkaloid biosynthesis in fruit.

A well-known defense-associated steroidal glycoalkaloid (SGA) metabolic shift eliminates the bitterness and toxicity of ripe tomato fruits. This study was conducted to clarify the effects of MADS-RIN (RIN) and its cofactors on SGA metabolism in tomato fruits. Using a CRISPR/Cas9-based gene-editing system, we mutated RIN and two cofactor genes (FUL1 and FUL2). The observed changes to fruit color and size in the mutants reflected the overlapping and distinct effects of RIN, FUL1, and FUL2 on fruit ripening. According to a UPLC-MS/MS analysis, the RIN and cofactor mutants had decreased levels of the relatively non-toxic metabolite esculeoside A, but they accumulated toxic SGA pathway intermediates, suggesting RIN and its cofactors are directly involved in esculeoside A biosynthesis. Transcriptome and qPCR analyses detected the downregulated expression of GAME5, which encodes a key enzyme mediating esculeoside A biosynthesis. ChIP-seq and ChIP-qPCR analyses confirmed GAME5 is targeted by RIN. RIN was observed to activate GAME5 transcription by binding to two non-canonical CArG-boxes in the GAME5 promoter. Additionally, RIN promotes SGA metabolism independently of ethylene. Collectively, these findings enhance our understanding of the molecular mechanism governing tomato fruit ripening and SGA biosynthesis. Furthermore, they may be useful for improving tomato fruit quality and safety.

The anti-tumor efficacy of a recombinant oncolytic herpes simplex virus mediated CRISPR/Cas9 delivery targeting in HPV16-positive cervical cancer

Cervical cancer, often driven by high-risk human papillomavirus (HPV) infections such as HPV16 or HPV18, remains a leading cause of cancer-related deaths. HPV16, found in about 90% of cervical cancer patients, harbors key oncogenic related genes (E6, E7, E2, E5) and an upstream regulatory region (URR) that contribute to cancer progression. This study introduces a novel approach using a recombinant oncolytic herpes simplex virus type 1 (HSV-1) named SONC103, armed with a CRISPR/Cas9 gene editing system. The aim was to target and disrupt integrated HPV16 genes in cervical cancer cells. Results demonstrated SONC103's capability to specifically and effectively knock down HPV16 oncogenes, thereby reducing cell proliferation and promoting apoptosis. Analyses further revealed loss of HPV16 DNA probes in infected cells' chromosomes, significant regulation of cellular processes related to tumor apoptosis, and downregulation of E6/E7 oncoproteins while increasing tumor suppressor proteins P53 and pRB. Notably, SONC103 exhibited substantial inhibition of tumor growth in a murine xenograft cervical cancer model. This study showcases the potential of the recombinant oncolytic HSV-1 virus (SONC103) in combating HPV16-positive cervical cancer by targeting oncogenes and facilitating oncolysis.

Cas9-PE: a robust multiplex gene editing tool for simultaneous precise editing and site-specific random mutation in rice

In molecular design breeding, the simultaneous introduction of desired functional genes through specific nucleotide modifications and the elimination of genes regulating undesired phenotypic traits or agronomic components require advanced gene editing tools. Due to limited editing efficiency, even with the use of highly precise editing tools, such as prime editing (PE), simultaneous editing of multiple mutation types poses a challenge. Here, we replaced Cas9 nickase (nCas9) with Cas9 to construct a Cas9-mediated PE (Cas9-PE) system in rice. This system not only enables precise editing, but also allows for site-specific random mutation. Moreover, leveraging the precision of Cas9-PE, we established a transgene-free multiplex gene editing system using a co-editing strategy. This strategy involved the Agrobacterium-mediated transient expression of the precise editing rice endogenous acetolactate synthase gene ALSS627I to confer herbicide bispyribac-sodium (BS) resistance as a selection marker. This study provides a versatile and efficient multiplex gene editing tool for molecular design breeding.

Modulating versatile pathways using a cleavable PEG shell and EGFR-targeted nanoparticles to deliver CRISPR-Cas9 and docetaxel for triple-negative breast cancer inhibition.

Human antigen R (HuR), an RNA-binding protein, is implicated in regulating mRNA stability and translation in cancer, especially in triple-negative breast cancer (TNBC), a highly aggressive form. CRISPR/Cas9-mediated HuR knockout (HuR CRISPR) presents a promising genetic therapeutic approach, but it encounters transfection limitations. Docetaxel (DTX), an effective cytotoxic agent against metastatic breast cancer (BC), faces challenges related to vehicle-associated adverse events in DTX formulations. Therefore, we designed multifunctional nanoparticles with pH-sensitive PEG derivatives and targeting peptides to enable efficient HuR CRISPR and DTX delivery to human TNBC MDA-MB-231 cells and tumor-bearing mice. Our findings indicated that these nanoparticles displayed pH-responsive cytotoxicity, precise EGFR targeting, efficient tumor penetration, successful endosomal escape, and accurate nuclear and cytoplasmic localization. They also demonstrated the ability to spare normal cells and prevent hemolysis. Our study concurrently modulated multiple pathways, including EGFR, Wnt/β-catenin, MDR, and EMT, through the regulation of EGFR/PI3K/AKT, HuR/galectin-3/GSK-3β/β-catenin, and P-gp/MRPs/BCRP, as well as YAP1/TGF-β/ZEB1/Slug/MMPs. The combined treatment arrested the cell cycle at the G2 phase and inhibited EMT, effectively impeding tumor progression. Tissue distribution, biochemical assays, and histological staining revealed the enhanced safety profile of pH-responsive PEG- and peptide-modified nanoformulations in TNBC mice. The DTX-embedded and peptide-modified nanoparticles mitigated the side effects of DTX, enhanced cytotoxicity in TNBC MDA-MB-231 cells, and exhibited remarkable antitumor efficacy and safety in TNBC-bearing mice with HuR CRISPR deletion. Collectively, the combination therapy of DTX and CRISPR/Cas9 offers an effective platform for delivering antineoplastic agents and gene-editing systems to combat tumor resistance and progression in TNBC.

Photocleavable Guide RNA for Photocontrolable CRISPR/Cas9 System

The development of gene editing systems on the base of CRISPR/Cas having higher efficacy, specificity, and possibility of their activity regulation by light irradiation is actually problem. Modification of CRISPR/Cas components, in particular guide RNA, by introduction of photocleavable linkers is the prospective approach for the solution of this problem. We developed the approach for the synthesis of photocleavable guide sgRNA for the CRISPR/Cas9 system containing the linkers on the base of 1-(2-nitrophenyl)-1,2-ethanediol. Such photomodified guide RNAs degrade upon UV-irradiation and CRISPR/Cas9 system is inactivated. We obtained three variants of photomodified sgRNA with different photolinker positions. It was demonstrated that sgRNA variant with photolinker introduced in the Cas9 protein binding and hairpins formation region is able to effectively guide Cas9 nuclease for DNA-target cleavage before UV-irradiation and lose its activity after irradiation. The conditions of controllable 40%-cleavage of model DNA-target were chosen. Developed approach provide specific inactivation of CRISPR/Cas9 gene editing system in specific time moment in definite place. Photoregulation of gene editing system permits not only reduce the undesirable off-target effects, but also becomes the basis of genetic disease therapy.

Therapeutic liver cell transplantation to treat murine PKU.

For gene therapy of the liver, in vivo applications based on adeno-associated virus are the most advanced vectors despite limitations, including low efficacy and episomal loss, potential integration and safety issues, and high production costs. Alternative vectors and/or delivery routes are of high interest. The regenerative ability of the liver bears the potential for ex vivo therapy using liver cell transplantation for disease correction if provided with a selective advantage to expand and replace the existing cell mass. Here we present such treatment of a mouse model of human phenylketonuria (PKU). Primary hepatocytes from wild-type mice were gene modified in vitro (with a lentiviral vector) that carries a gene editing system (CRISPR) to inhibit Cypor. Cypor inactivation confers paracetamol (or acetaminophen) resistance to hepatocytes and thus a growth advantage to eliminate the pre-existing liver cells upon grafting (via the spleen) and exposure to repeated treatment with paracetamol. Grafting Cypor-inactivated wild-type hepatocytes into inbred young adult enu2 (PKU) mice, followed by selective expansion by paracetamol dosing, resulted in replacing up to 5% of cell mass, normalization of blood phenylalanine, and permanent correction of PKU. Hepatocyte transplantation offers thus an armamentarium of novel therapy options for genetic liver defects.

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.

Genetic manipulation of the genes for clonal seeds results in sterility in cotton

BackgroundHeterosis is a common phenomenon in plants and has been extensively applied in crop breeding. However, the superior traits in the hybrids can only be maintained in the first generation but segregate in the following generations. Maintaining heterosis in generations has been challenging but highly desirable in crop breeding. Recent study showed that maternally produced diploid seeds could be achieved in rice by knocking out three meiosis related genes, namely REC8, PAIR1, OSD1 to create MiMe in combination with egg cell specific expression of BBM transcription factor, a technology called clonal seeds. Interestingly, there has been very limited reports indicating the feasibility of this approach in other crops.ResultsIn this study, we aimed to test whether clonal seeds could be created in cotton. We identified the homologs of the three meiosis related genes in cotton and used the multiplex CRISPR/Cas9 gene editing system to simultaneously knock out these three genes in both A and D sub-genomes. More than 50 transgenic cotton plants were generated, and fragment analysis indicated that multiple gene knockouts occurred in the transgenic plants. However, all the transgenic plants were sterile apparently due to the lack of pollen. Pollination of the flowers of the transgenic plants using the wild type pollens could not generate seeds, an indication of defects in the formation of female sexual cells in the transgenic plants. In addition, we generated transgenic cotton plants expressing the cotton BBM gene driven by the Arabidopsis egg cell specific promoter pDD45. Two transgenic plants were obtained, and both showed severely reduced fertility.ConclusionsOverall, our results indicate that knockout of the clonal seeds related genes in cotton causes sterility and how to manipulate genes to create clonal seeds in cotton requires further research.

Allelic Variation of BnaFTA2 and BnaFTC6 Is Associated With Flowering Time and Seasonal Crop Type in Rapeseed (Brassica napus L.).

Different ecological types of rapeseed (Brassica napus L.), including winter, spring, and semi-winter cultivars, exhibit varying flowering times and cannot be planted in the same cultivation areas. FLOWERING LOCUS T (FT) plays a key role in regulating flowering. In allotetraploid B. napus six copies of FT (BnaFT) have been reported. However, there is uncertainty about how the translated products of each paralog, as well as cis-allelic variations at each locus, contribute functionally to flowering time and define specific crop types. In this study, we confirm that BnaFT exhibit distinct expression patterns in different crop types of rapeseed. Using the CRISPR/Cas9 gene editing system, we provide functional evidence that the mutants between Bnaft paralogues affects the regulation of flowering time. Furthermore, we identify a new haplotype of BnaFT.A2 that is associated with early flowering time, although this appears necessary but not sufficient to confer a spring type phenotype. Three haplotypes of BnaFT.C6 were further identified and associated with both flowering time and crop types. We speculate that variations in both BnaFT.A2 and BnaFT.C6 may have undergone diversifying selection during the divergence of seasonal crop types in rapeseed.

A biodegradable lipid nanoparticle delivers a Cas9 ribonucleoprotein for efficient and safe in situ genome editing in melanoma

The development of melanoma is closely related to Braf gene, which is a suitable target for CRISPR/Cas9 based gene therapy. CRISPR/Cas9-sgRNA ribonucleoprotein complexes (RNPs) stand out as the safest format compared to plasmid and mRNA delivery. Similarly, lipid nanoparticles (LNPs) emerge as a safer alternative to viral vectors for delivering the CRISPR/Cas9-sgRNA gene editing system. Herein, we have designed multifunctional cationic LNPs specifically tailored for the efficient delivery of Cas9 RNPs targeting the mouse Braf gene through transdermal delivery, aiming to treat mouse melanoma. LNPs are given a positive charge by the addition of a newly synthesized polymer, deoxycholic acid modified polyethyleneimine (PEI-DOCA). Positive charge enables LNPs to be delivered in vivo by binding to negatively charged cell membranes and proteins, thereby facilitating efficient skin penetration and enhancing the delivery of RNPs into melanoma cells for gene editing purposes. Our research demonstrates that these LNPs enhance drug penetration through the skin, successfully delivering the Cas9 RNPs system and specifically targeting the Braf gene. Cas9 RNPs loaded LNPs exert a notable impact on gene editing in melanoma cells, significantly suppressing their proliferation. Furthermore, in mice experiments, the LNPs exhibited skin penetration and tumor targeting capabilities. This innovative LNPs delivery system offers a promising gene therapy approach for melanoma treatment and provides fresh insights into the development of safe and effective delivery systems for Cas9 RNPs in vivo. Statement of significanceCRISPR/Cas9 technology brings new hope for cancer treatment. Cas9 ribonucleoprotein offers direct genome editing, yet delivery challenges persist. For melanoma, transdermal delivery minimizes toxicity but faces skin barrier issues. We designed multifunctional lipid nanoparticles (LNPs) for Cas9 RNP delivery targeting the Braf gene. With metal microneedle pretreatment, our LNPs effectively edited melanoma cells, reducing Braf expression and inhibiting tumor growth. Our study demonstrates LNPs' potential for melanoma therapy and paves the way for efficient in vivo Cas9 RNP delivery systems in cancer therapy.

Gene Editing with Artificial DNA Scissors.

Artificial metallo-nucleases (AMNs) are small molecule DNA cleavage agents, also known as DNA molecular scissors, and represent an important class of chemotherapeutic with high clinical potential. This review provides a primary level of exploration on the concepts key to this area including an introduction to DNA structure, function, recognition, along with damage and repair mechanisms. Building on this foundation, we describe hybrid molecules where AMNs are covalently attached to directing groups that provide molecular scissors with enhanced or sequence specific DNA damaging capabilities. As this research field continues to evolve, understanding the applications of AMNs along with synthetic conjugation strategies can provide the basis for future innovations, particularly for designing new artificial gene editing systems.

Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenic Phytophthora species.

Plasmid-mediated DNA transformation is a foundational molecular technique and the basis for most CRISPR-Cas9 gene editing systems. While plasmid transformations are well established for many agricultural Phytophthora pathogens, development of this technique in forest Phytophthoras is lacking. Given our long-term research objective to develop CRISPR-Cas9 gene editing in a forest pathogenic Phytophthora species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: P. cactorum, P. cinnamomi, P. cryptogea, P. ramorum, and P. syringae. We used the agricultural pathogen P. sojae, a species for which PEG-mediated transformations are well-established, as a transformation control. Using a protocol previously optimized for P. sojae, we tested transformations in the five forest Phytophthoras with three different plasmids: two developed for CRISPR-Cas9 gene editing and one developed for fluorescent protein tagging. Out of the five species tested, successful transformation, as indicated by stable growth of transformants on a high concentration of antibiotic selective growth medium and diagnostic PCR, was achieved only with P. cactorum and P. ramorum. However, while transformations in P. cactorum were consistent and stable, transformations in P. ramorum were highly variable and yielded transformants with very weak mycelial growth and abnormal morphology. Our results indicate that P. cactorum is the best candidate to move forward with CRISPR-Cas9 protocol development and provide insight for future optimization of plasmid transformations in forest Phytophthoras.

Exploration of the pneumocandin biosynthetic gene cluster based on efficient CRISPR/Cas9 gene editing strategy in Glarea lozoyensis

Pneumocandin B0 (PB0) is a lipopeptide produced by the fungus Glarea lozoyensis. The existing challenges with the low-yield and the extended-fermentation cycle emphasize necessity for strain improvement. In this study, we optimized conditions to obtain high-quality protoplasts and screened effective selection markers, leading to the construction of three CRISPR/Cas9 gene editing systems. Utilizing a constitutive Cas9 expression recipient strain, combined with dual sgRNAs targeting, we achieved highly efficient editing of target genes. We successfully knocked out 10 genes within the pneumocandin putative biosynthetic gene cluster and analyzed their roles in PB0 production. Our findings reveal that 4 of 10 genes are directly involved in PB0 production. Specially, the deletion of gltrt or gl10050 resulted in reduced PB0 production, while the absence of glhyp or glhtyC led to the complete loss of PB0 biosynthesis. Notably, the deletion of glhyp caused the silencing of nearly all cluster genes, whereas overexpression of glhyp led to a 2.38-fold increase in PB0 production. Therefore, this study provides the first comprehensive exploration of the functions of 10 genes within the pneumocandin putative biosynthetic gene cluster. Our findings provide valuable technical strategies for constructing bioengineering strains with purposefully enhanced PB0 production.

Brain-Targeted Cas12a Ribonucleoprotein Nanocapsules Enable Synergetic Gene Co-Editing Leading to Potent Inhibition of Orthotopic Glioblastoma.

Gene-editing technology shows great potential in glioblastoma (GBM) therapy. Due to the complexity of GBM pathogenesis, a single gene-editing-based therapy is unlikely to be successful; therefore, a multi-gene knockout strategy is preferred for effective GBM inhibition. Here, a non-invasive, biodegradable brain-targeted CRISPR/Cas12a nanocapsule is used that simultaneously targeted dual oncogenes, EGFR and PLK1, for effective GBM therapy. This cargo nanoencapsulation technology enables the CRISPR/Cas12a system to achieve extended blood half-life, efficient blood-brain barrier (BBB) penetration, active tumor targeting, and selective release. In U87MG cells, the combinatorial gene editing system resulted in 61% and 33% knockout of EGFR and PLK1, respectively. Following systemic administration, the CRISPR/Cas12a system demonstrated promising brain tumor accumulation that led to extensive EGFR and PLK1 gene editing in both U87MG and patient-derived GSC xenograft mouse models with negligible off-target gene editing detected through NGS. Additionally, CRISPR/Cas12a nanocapsules that concurrently targeted the EGFR and PLK1 oncogenes showed superior tumor growth suppression and significantly improved the median survival time relative to nanocapsules containing single oncogene knockouts, signifying the potency of the multi-oncogene targeting strategy. The findings indicate that utilization of the CRISPR/Cas12a combinatorial gene editing technique presents a practical option for gene therapy in GBM.

Gene editing by SSB/CRISPR-Cas9 ribonucleoprotein in bacteria

The application of CRISPR-Cas9 ribonucleoprotein (RNP) for gene editing is commonly used in plants and animals, but its application in bacteria has not been reported. In this study, we employed DNA single-strand binding protein (SSB) to construct an SSB/CRISPR-Cas9 RNP-editing system for non-homologous recombination and homologous recombination gene editing of the upp gene in bacteria. The RNP targeting the upp gene, along with SSB, was introduced into the protoplasts of Escherichia coli, Pseudomonas, and Bacillus subtilis. Transformants were obtained on plates containing 5-fluorouracil (5-FU) with gene editing efficiencies (percentage of transformants relative to the number of protoplasts) of 9.75 %, 5.02 %, and 8.37 %, respectively, and sequencing analysis confirmed 100 % non-homologous recombination. When RNP, SSB, and a 100-nucleotide single-stranded oligodeoxynucleotide (ssODN) donor were introduced into the protoplasts of these bacteria, transformants were obtained with editing efficiencies of 45.11 %, 30.13 %, and 27.18 %, respectively, and sequencing confirmed 100 % homologous recombination knockout of the upp gene. Additionally, introducing RNP, SSB, and a 100 base-pair double-stranded oligodeoxynucleotide (dsODN) donor containing a tetracycline resistance gene (tetR-dsODN) resulted in transformants on 5-FU plates with editing efficiencies of 35.94 %, 22.46 %, and 19.08 %, respectively, with sequencing confirming 100 % homologous recombination replacement of the upp gene with tetR. These results demonstrate that the SSB/CRISPR-Cas9 RNP system can efficiently, simply, and rapidly edit bacterial genomes without the need for plasmids. This study is the first to report the use of RNP-based gene editing in bacteria.