Gene Editing Efficiency Research Articles

The Influence of Homologous Arm Length on Homologous Recombination Gene Editing Efficiency Mediated by SSB/CRISPR-Cas9 in Escherichia coli.

The precise editing of genes mediated by CRISPR-Cas9 necessitates the application of donor DNA with appropriate lengths of homologous arms and fragment sizes. Our previous development, SSB/CRISPR-Cas9, has demonstrated high efficiency in homologous recombination and non-homologous end joining gene editing within bacteria. In this study, we optimized the lengths and sizes of homologous arms of the donor DNA within this system. Two sets of donor DNA constructs were generated: one set comprised donors with only 10-100 bp homologous arms, while the other set included donors with homologous arms ranging from 10-100 bp, between which was a tetracycline resistance expression cassette (1439 bp). These donor constructs were transformed into Escherichia coli MG1655 cells alongside pCas-SSB/pTargetF-lacZ. Notably, when the homologous arms ranged from 10 to 70 bp, the transformation efficiency of non-selectable donors was significantly higher than that of selectable donors. However, within the range of 10-100 bp homologous arm lengths, the homologous recombination rate of selectable donors was significantly higher than that of non-selectable donors, with the gap narrowing as the homologous arm length increased. For selectable donor DNA with homologous arm lengths of 10-60 bp, the homologous recombination rate increased linearly, reaching a plateau when the homologous arm length was between 60-100 bp. Conversely, for non-selectable donor DNA, the homologous recombination rate increased linearly with homologous arm lengths of 10-90 bp, plateauing at 90-100 bp. Editing two loci simultaneously with 100 bp homologous arms, whether selectable or non-selectable, showed no difference in transformation or homologous recombination rates. Editing three loci simultaneously with 100 bp non-selectable homologous arms resulted in a 45% homologous recombination rate. These results suggest that efficient homologous recombination gene editing mediated by SSB/CRISPR-Cas9 can be achieved using donor DNA with 90-100 bp non-selectable homologous arms or 60-100 bp selectable homologous arms.

Establishment and characterization of Lesch-Nyhan syndrome rabbit model.

Lesch-Nyhan syndrome (LNS) is a congenital defect disease that results in defective purine metabolism. It is caused by pathogenic variants of the HPRT gene. Its clinical symptoms mainly include high uric acid levels, gout, and kidney stones and damage. The mechanism of LNS has not been fully elucidated, and no cure exists. Animal models have always played an important role in exploring causative mechanisms and new therapies. This study combined CRISPR/Cas9 and microinjection to knock out the HPRT gene to create an LNS rabbit model. A sgRNA targeting exon 3 of HPRT gene was designed. Subsequently, Cas9 mRNA and sgRNA were injected into rabbit zygotes, and injected embryos were transferred to the uterus. The genotype and phenotype of rabbits were analyzed after birth. Four infant rabbits (named R1, R2, R3 and R4), which showed varying levels of gene modification, were born. The gene-editing efficiency was 100%. No wild-type sequences at the target HPRT gene were detected in R4 rabbit. Next, 6-thioguanine drug testing confirmed that HPRT enzymatic activity was deficient in R4 infant rabbit. HE staining revealed kidney abnormalities in all infant rabbits. Overall, an sgRNA capable of knocking out the HPRT gene in rabbits was successfully designed, and HPRT gene-modified rabbits were successfully constructed by using CRISPR/Cas9 technology and microinjection. This study provides a new nonrodent animal model for studying LNS syndrome.

O17 Highly efficient base editing of homozygous and heterozygous COL7A1 variants in fibroblasts from patients with recessive dystrophic epidermolysis bullosa using adenine base editor 8e mRNA system delivered via novel lipid nanoparticle formulations

Abstract Introduction and aims Adenine base editors (ABEs) present an attractive approach to gene editing in the inherited blistering disease recessive dystrophic epidermolysis bullosa (RDEB), which Results from pathogenic variants in COL7A1. We previously showed that ABE8e, a new ABE variant, confers remarkable efficiency (94.6%) and a favourable safety profile in correcting variants and restoring collagen 7 (C7) expression when electroporated into RDEB fibroblasts. With a goal to develop topical base editing creams, we now explore use of lipid nanoparticle (LNP)-encapsulated ABE8e mRNA system to edit two distinct variants in RDEB fibroblasts. Methods Primary patient fibroblasts harbouring a homozygous G>A mutation (exon 42) or a heterozygous C>T mutation (exon 54) were gene edited using ABE8e mRNA, delivered via lipofectamine or six different LNPs. To assess the gene editing efficiency, Sanger sequencing and next-generation sequencing were employed. Cell viability was evaluated via a lactate dehydrogenase cytotoxicity assay, while functional restoration of C7 expression was assessed via Western blotting of cell lysate and supernatant. Finally, topical delivery was tested on normal human skin equivalents (HSEs). Results We identified an optimal ratio of 1 : 7 for sgRNA : ABE8e, at which editing efficiency increases with ABE8e dosage, with 400 ng being sufficient for 90% biallelic editing. Formulations containing DTDTMA (C14) and DHDTMA (C16) LNPs demonstrated approximately 90% and approximately 80% editing (with and without the peptide, respectively), in addition to reduced cytotoxicity compared with lipofectamine. Depending on the nature of the mutation, the edit corresponded to an increase in C7 expression in supernatant only or cell lysate as well, whereby C14 LNPs enabled twofold C7 restoration. Finally, our HSEs demonstrated viability of topical delivery of lipofectamine-encapsulated green fluorescent protein mRNA. Conclusions We demonstrated the potential value of base editing as a gene therapy for RDEB in addition to the potential of these LNPs for clinical translation of the therapy. Nonetheless, further safety and functionality tests still need to be conducted.

Increasing the efficiency of gene editing with CRISPR-Cas9 via concurrent expression of the Beta protein

Escherichia coli has emerged as an important host for the production of biopharmaceuticals or other industrially relevant molecules. An efficient gene editing tool is indispensable for ensuring high production levels and optimal release of target products. However, in Escherichia coli, the CRISPR-Cas9 system has been shown to achieve gene modifications with relatively low frequency. Large-scale PCR screening is required, hindering the identification of positive clones. The beta protein, which weakly binds to single-stranded DNA but tightly associates with complementary strand annealing products, offers a promising solution to this issue. In the present study, we describe a targeted and continuous gene editing strategy for the Escherichia coli genome. This strategy involves the coexpression of the beta protein alongside the CRISPR-Cas9 system, enabling a variety of genome modifications such as gene deletion and insertion with an efficiency exceeding 80 %. The integrity of beta proteins is essential for the CRISPR-Cas9/Beta-based gene editing system. In this work, the deletion of either the N- or C-terminal domain significantly impaired system efficiency. Overall, our findings established the CRISPR-Cas9/Beta system as a suitable gene editing tool for various applications in Escherichia coli.

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways

Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

Triterpenoids-templated self-assembly nanosystem for biomimetic delivery of CRISPR/Cas9 based on the synergy of TLR-2 and ICB to enhance HCC immunotherapy

Combination immunotherapy has shown promising potential for enhancing the objective response rate compared to immune checkpoint blockade (ICB) monotherapy. However, combination therapy with multi-drugs is limited by the different properties of the agents and inconsistent synergistic targeted delivery. Herein, based on a universal triterpene template and the anticancer active agent ursolic acid (UA), a cytomembrane-coated biomimetic delivery nanoplatform (UR@M) prepared by the self-assembly of a PD-L1 targeted CRISPR/Cas9 system and UA was designed for hepatocellular carcinoma (HCC) treatment. UR@M showed enhanced tumor accumulation in vivo with homologous tumor targeting, and CRISPR in the nanosystem exhibited potent gene-editing efficiency of 76.53% in vitro and 62.42% in vivo with no off-target effects. UA activated the natural immune system through the TLR-2-MyD88-TRAF6 pathway, which synergistically enhanced the proliferation of natural killer cells and dendritic cells and realized excellent immune cytotoxic T cell infiltration by combining with the ICB of PD-L1. The strategy of work along both lines based on innate immune and adaptive immunity displayed a significant effect in tumor regression. Overall, the UA-templated strategy “killed three birds with one stone” by establishing a self-assembly nanosystem, inducing tumor cell death, and promoting synergistic immunostimulation for HCC treatment.

Increasing CRISPR/Cas9-mediated gene editing efficiency in T7 phage by reducing the escape rate based on insight into the survival mechanism.

Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages. However, some bacteriophages can escape from the cleavage of Cas protein, such as Cas9, and decrease the efficiency of genome editing. This study focuses on the bacteriophage T7, which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated. First, we test the escape rates of T7 phage at different cleavage sites, ranging from 10 -2 to 10 -5. The sequencing results show that DNA point mutations and microhomology-mediated end joining (MMEJ) at the target sites are the main causes. Next, we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region. Moreover, we also knock out the ATP-dependent DNA ligase 1. 3 gene, which may be involved in the MMEJ event, and the frequency of MMEJ at 4. 3 is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ 1. 3 increases from 20% to 100%. This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1. 3 can reduce MMEJ events and enhance gene editing efficiency. These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.

Recent Therapeutic Gene Editing Applications to Genetic Disorders.

Recent years have witnessed unprecedented progress in therapeutic gene editing, revolutionizing the approach to treating genetic disorders. In this comprehensive review, we discuss the progression of milestones leading to the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)-based technology as a powerful tool for precise and targeted modifications of the human genome. CRISPR-Cas9 nuclease, base editing, and prime editing have taken center stage, demonstrating remarkable precision and efficacy in targeted ex vivo and in vivo genomic modifications. Enhanced delivery systems, including viral vectors and nanoparticles, have further improved the efficiency and safety of therapeutic gene editing, advancing their clinical translatability. The exploration of CRISPR-Cas systems beyond the commonly used Cas9, such as the development of Cas12 and Cas13 variants, has expanded the repertoire of gene editing tools, enabling more intricate modifications and therapeutic interventions. Outstandingly, prime editing represents a significant leap forward, given its unparalleled versatility and minimization of off-target effects. These innovations have paved the way for therapeutic gene editing in a multitude of previously incurable genetic disorders, ranging from monogenic diseases to complex polygenic conditions. This review highlights the latest innovative studies in the field, emphasizing breakthrough technologies in preclinical and clinical trials, and their applications in the realm of precision medicine. However, challenges such as off-target effects and ethical considerations remain, necessitating continued research to refine safety profiles and ethical frameworks.

Increasing Knockin Efficiency in Mouse Zygotes by Transient Hypothermia.

Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates. Using a traffic light reporter in mouse embryonic stem cells and a fluorescence conversion reporter in human-induced pluripotent stem cells, we confirm that a transient cold shock leads to an increase in the rate of HDR, with a corresponding decrease in the rate of NHEJ repair. We then investigated whether a similar cold shock could lead to an increase in the rate of HDR in the mouse embryo. By analyzing the efficiency of gene editing using single nucleotide polymorphism changes and loxP insertion at three different genetic loci, we found that a transient reduction in temperature after zygote electroporation of CRISPR-Cas9 ribonucleoprotein with a single-stranded oligodeoxynucleotide repair template did indeed increase knockin efficiency, without affecting embryonic development. The efficiency of gene editing with and without the cold shock was first assessed by genotyping blastocysts. As a proof of concept, we then confirmed that the modified embryo culture conditions were compatible with live births by targeting the coat color gene tyrosinase and observing the repair of the albino mutation. Taken together, our data suggest that a transient cold shock could offer a simple and robust way to improve knockin outcomes in both stem cells and zygotes.

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.

CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200µm and a spacing of 150µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.

A Novel CRISPR/Cas9‐Encapsulated Biomimetic Manganese Sulfide Nanourchins for Targeted Magnetic Resonance Contrast Enhancement and Self‐Enhanced Chemodynamics‐Gene‐Immune Synergistic Tumor Therapy

AbstractCRISPR/Cas9 gene editing systems are widely studied in cancer therapy, however, it is still challenging to improve the gene editing efficiency and enhance the anticancer efficacy. Herein, a novel self‐assembled manganese sulfide nanourchin with CRISPR/Cas9 and hybrid membrane encapsulation (MCRT) is developed to highlight the synergistic mechanism of hydrogen sulfide (H2S) and CRISPR/Cas9 in cancer therapy. The special structure and cationic surface of nanourchins facilitate the loading of CRISPR/Cas9 with the large size and the encapsulation of erythrocyte‐tumor cell hybrid membrane, endowing the nanoplatform with long circulation and precise tumor targeting. MCRT with pH‐response can effectively decompose into H2S and Mn2+, and release CRISPR/Cas9 in acidic tumor microenvironment. H2S can up‐regulate intracellular hydrogen peroxide level by inhibiting the activity of catalase, which enables self‐enhanced chemodynamic therapy (CDT) of Mn2+. In addition, H2S can synergize with the released CRISPR/Cas9, co‐downregulating the level of intracellular anti‐apoptotic protein survivin, achieving self‐enhanced gene therapy. Furthermore, systemic delivery of MCRT displays significantly tumor magnetic resonance imaging (MRI) contrast enhancement. Importantly, H2S‐enhanced CDT‐gene therapy simultaneously induces immunogenic cell death and triggered a robust anti‐tumor immune response. Thus, MCRT has a potential application for the targeted MRI and self‐enhanced CDT‐gene‐immune synergistic tumor therapy.

AI-driven Protein Engineering for DNA Sequence Modification

The integration of artificial intelligence (AI) with gene editing technologies like CRISPR-Cas9 holds immense promise for advancing biomedical research and personalized medicine. This article highlights the crucial role of AI in predicting and minimizing off-target effects, thereby enhancing the precision and efficiency of gene editing. Researchers have developed algorithms like BE-DICT to accurately predict base editing outcomes, showcasing the potential of AI-driven strategies in optimizing gene editing processes. By combining AI with bioengineering, this interdisciplinary approach aims to automate and refine DNA modifications, paving the way for innovative applications in personalized gene therapy and biofabrication. Ultimately, this research endeavors to revolutionize the life sciences field, leading to significant breakthroughs in healthcare and biotechnology.

Metabolic engineering of the probiotic Escherichia coli Nissle 1917 for lacto-N-triose II production

Escherichia coli Nissle 1917 (EcN) is a probiotic used to treat gastrointestinal diseases. In recent years, the development of metabolic engineering has promoted the application of EcN in synthetic biology. Lacto-N-triose II (LNT II), as an important component in breast milk, has unique benefits for infant health. This study introduces a metabolic engineering method to produce LNT II in EcN. Firstly, construct promoters with different expression intensities to optimize transcriptional regulation levels and further fine-tune plasmid copy numbers to optimize gene expression levels. The highest titer of LNT II was detected when the lgtA gene was expressed under the tac promoter and pETDuet-1 vector. Secondly, the gene editing efficiency of the EcN strain was improved by knocking out the endA gene (encoding the endonuclease I). Then, a series of engineered strains were constructed by knocking out the wecB gene and cryptic plasmid. It was found that the deletion of the wecB gene increased the titer of LNT II, but the elimination of the cryptic plasmid had no effect. Finally, we validated the production performance of the engineering strain E13 in a 3-L bioreactor with batch feeding culture. The LNT II titer reached 2040 mg/L, which was 6.6 times higher than that of shake flask cultivation and has potential for industrial application.

New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i

BackgroundFor ten years, CRISPR/cas9 system has become a very useful tool for obtaining site-specific mutations on targeted genes in many plant organisms. This technology opens up a wide range of possibilities for improved plant breeding in the future. In plants, the CRISPR/Cas9 system is mostly used through stable transformation with constructs that allow for the expression of the Cas9 gene and sgRNA. Numerous studies have shown that site-specific mutation efficiency can vary greatly between different plant species due to factors such as plant transformation efficiency, Cas9 expression, Cas9 nucleotide sequence, the addition of intronic sequences, and many other parameters. Since 2016, when the first edited grapevine was created, the number of studies using functional genomic approaches in grapevine has remained low due to difficulties with plant transformation and gene editing efficiency. In this study, we optimized the process to obtain site-specific mutations and generate knock-out mutants of grapevine (Vitis vinifera cv. ‘Chardonnay’). Building on existing methods of grapevine transformation, we improved the method for selecting transformed plants at chosen steps of the developing process using fluorescence microscopy.ResultsBy comparison of two different Cas9 gene and two different promoters, we increased site-specific mutation efficiency using a maize-codon optimized Cas9 containing 13 introns (zCas9i), achieving up to 100% biallelic mutation in grapevine plantlets cv. ‘Chardonnay’. These results are directly correlated with Cas9 expression level.ConclusionsTaken together, our results highlight a complete methodology for obtaining a wide range of homozygous knock-out mutants for functional genomic studies and future breeding programs in grapevine.

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

BackgroundCRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources.MethodsWe screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β).ResultsWe established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions.ConclusionsWe showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.

Streamlined Agrobacterium rhizogenes-mediated hairy root transformation for efficient CRISPR/Cas9-based gene editing evaluation in diverse Citrullus varieties

The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics. Within this system, single guide RNAs (sgRNAs) are instrumental in the precise cleavage of DNA double strands. However, the efficiency of gene editing varies among sgRNAs, emphasizing the need to meticulously select target sites, especially in the context of Citrullus lanatus, a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation. This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species. Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus, Citrullus mucosospermus, and Citrullus amarus. Employing a vector with two sgRNAs (sgRNA1 and sgRNA5) positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system, targeted mutations were detected in 90.9% of accessions across the four Citrullus species. Notably, 73.94% of all examined hairy roots exhibited mutations at the sgRNA1 site, while the sgRNA5 site showed no mutations. Among the 31 different mutation types identified at the sgRNA1 site, base deletion was the most prevalent. Using the sgRNA1 site of ClCIPK17, stable transgenic watermelon buds were obtained from explants, and the targeted mutations of the sgRNA1 site were confirmed. These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.

A method of genetic transformation and gene editing of succulents without tissue culture.

Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties - the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%-7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.

Biologically produced and metal-organic framework delivered dual-cut CRISPR/Cas9 system for efficient gene editing and sensitized cancer therapy

Manipulation of the lactate metabolism is an efficient way for cancer treatment given its involvement in cancer development, metastasis, and immune escape. However, most of the inhibitors of lactate transport carriers suffer from poor specificity. Herein, we use the CRISPR/Cas9 system to precisely downregulate the monocarboxylate carrier 1 (MCT1) expression. To avoid the self-repairing during the gene editing process, a dual-Cas9 ribonucleoproteins (duRNPs) system is generated using the biological fermentation method and delivered into cells by the zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, enabling precise removal of a specific DNA fragment from the genome. For efficient cancer therapy, a specific glucose transporter 1 inhibitor (BAY-876) is co-delivered with the duRNPs, forming BAY/duRNPs@ZIF-8 nanoparticle. ZIF-8 nanoparticles can deliver the duRNPs into cells within 1 h, which efficiently downregulates the MCT1 expression, and prohibits lactate influx. Through simultaneous inhibition of the lactate and glucose influx, BAY/duRNPs@ZIF-8 prohibits ATP generation, arrests cell cycle, inhibits cell proliferation, and finally induces cellular apoptosis both in vitro and in vivo. Consequently, we demonstrate that the biologically produced duRNPs delivered into cells by the nonviral ZIF-8 carrier have expanded the CRISPR/Cas gene editing toolbox and elevated the gene editing efficiency, which will promote biological studies and clinical applications. Statement of significanceThe CRISPR/Cas9 system, widely used as an efficient gene editing tool, faces a challenge due to cells’ ability to self-repair. To address this issue, a strategy involving dual-cutting of the genome DNA has been designed and implemented. This strategy utilizes biologically produced dual-ribonucleoproteins delivered by a metal-organic framework. The effectiveness of this dual-cut CRISPR-Cas9 system has been demonstrated through a therapeutic approach targeting the simultaneous inhibition of lactate and glucose influx in cancer cells. The utilization of the dual-cut gene editing strategy has provided valuable insights into gene editing and expanded the toolbox of the CRISPR/Cas-based gene editing system. It has the potential to enable more efficient and precise manipulation of specific protein expression in the future.

Improving the Efficiency of CRISPR Ribonucleoprotein-Mediated Precise Gene Editing by Small Molecules in Porcine Fibroblasts.

The aim of this study was to verify whether small molecules can improve the efficiency of precision gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoprotein (RNP) in porcine cells. CRISPR associated 9 (Cas9) protein, small guide RNA (sgRNA), phosphorothioate-modified single-stranded oligonucleotides (ssODN), and different small molecules were used to generate precise nucleotide substitutions at the insulin (INS) gene by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by polymerase chain reaction (PCR) for the target site. All samples were sequenced and analyzed, and the efficiencies of different small molecules at the target site were compared. The results showed that the optimal concentrations of the small molecules, including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A, for in vitro-cultured PFFs' viability were determined. Compared with the control group, the single small molecules including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A increased the efficiency of HDR-mediated precise gene editing from 1.71-fold to 2.28-fold, respectively. There are no benefits in using the combination of two small molecules, since none of the combinations improved the precise gene editing efficiency compared to single small molecules. In conclusion, these results suggested that a single small molecule can increase the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells.

Iron-Confined CRISPR/Cas9-Ribonucleoprotein Delivery System for Redox-Responsive Gene Editing.

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) is a promising gene editing tool to treat diseases at the genetic level. Nonetheless, the challenge of the safe and efficient delivery of CRISPR/Cas9 to host cells constrains its clinical applicability. In the current study, a facile, redox-responsive CRISPR/Cas9-Ribonucleoprotein (RNP) delivery system by combining iron-coordinated aggregation with liposomes (Fe-RNP@L) is reported. The Fe-RNP is formed by the coordination of Fe3+ with amino and carboxyl groups of Cas9, which modifies the lipophilicity and surface charge of RNP and alters cellular uptake from primary endocytosis to endocytosis and cholesterol-dependent membrane fusion. RNP can be rapidly and reversibly released from Fe-RNP in response to glutathione without loss of structural integrity and enzymatic activity. In addition, iron coordination also improves the stability of RNP and substantially mitigates cytotoxicity. This construct enabled highly efficient cytoplasmic/nuclear delivery (≈90%) and gene-editing efficiency (≈70%) even at low concentrations. The high payload content, high editing efficiency, good stability, low immunogenicity, and ease of production and storage, highlight its potential for diverse genome editing and clinical applications.