Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.     

Harnessing the Potential of CRISPR/Cas in Atherosclerosis: Disease Modeling and Therapeutic Applications

Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.     

Intracellular Delivery of mRNA for Cell-Selective CRISPR/Cas9 Genome Editing using Lipid Nanoparticles

Messenger RNA (mRNA) is being used as part of an emerging class of biotherapeutics with great promise for preventing and treating a wide range of diseases, as well as encoding programmable nucleases for genome editing. However, mRNA's low stability and immunogenicity, as well as the impermeability of the cell membrane to mRNA greatly limit mRNA's potential for therapeutic use. Lipid nanoparticles (LNPs) are currently one of the most extensively studied nanocarriers for mRNA delivery and have recently been clinically approved for developing mRNA-based vaccines to prevent COVID-19. In this review, we summarize the latest advances in designing ionizable lipids and formulating LNPs for intracellular and tissue-targeted mRNA delivery. Furthermore, we discuss the progress of intracellular mRNA delivery for spatiotemporally controlled CRISPR/Cas9 genome editing by using LNPs. Finally, we provide a perspective on the future of LNP-based mRNA delivery for CRISPR/Cas9 genome editing and the treatment of genetic disorders.     

Stimulus-Responsive Smart Nanoparticles-Based CRISPR-Cas Delivery for Therapeutic Genome Editing

The innovative research in genome editing domains such as CRISPR-Cas technology has enabled genetic engineers to manipulate the genomes of living organisms effectively in order to develop the next generation of therapeutic tools. This technique has started the new era of “genome surgery”. Despite these advances, the barriers of CRISPR-Cas9 techniques in clinical applications include efficient delivery of CRISPR/Cas9 and risk of off-target effects. Various types of viral and non-viral vectors are designed to deliver the CRISPR/Cas9 machinery into the desired cell. These methods still suffer difficulties such as immune response, lack of specificity, and efficiency. The extracellular and intracellular environments of cells and tissues differ in pH, redox species, enzyme activity, and light sensitivity. Recently, smart nanoparticles have been synthesized for CRISPR/Cas9 delivery to cells based on endogenous (pH, enzyme, redox specie, ATP) and exogenous (magnetic, ultrasound, temperature, light) stimulus signals. These methodologies can leverage genome editing through biological signals found within disease cells with less off-target effects. Here, we review the recent advances in stimulus-based smart nanoparticles to deliver the CRISPR/Cas9 machinery into the desired cell. This review article will provide extensive information to cautiously utilize smart nanoparticles for basic biomedical applications and therapeutic genome editing.     

Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.     

New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.     

Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology—several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcus pyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.     

Dibenzocyclooctyne-Branched Primer Assembled Gene Nanovector and Its Potential Applications in Genome Editing

The CRISPR/Cas9 system has been widely used as an efficient genome editing toolkit for gene therapy. The delivery of vectors encoding the full CRISPR/Cas9 components including Cas9 gene and gRNA expression element into cells is the crucial step to effective genome editing. However, the cargo gene sequence for genome editing is usually large, which reduces the cargo encapsulation efficiency and affects the vector size. To obtain a nanovector with high cargo gene loading capacity and biocompatible size, we report the construction of a gene nanovector from branch-PCR with a dibenzocyclooctyne (DBCO)-branched primer and establish the correlation mapping between gene length and nanovector size. The results show that the size of nanovectors can be tuned according to the gene length. According to the findings, we constructed nanovectors carrying the full CRISPR/Cas9 components in 100–200 nm and validated their application in genome editing. The results show that this kind of nanovector exhibits higher serum stability than plasmids and can reach comparable genome editing efficiency with plasmids. Hence, this type of gene nanovector obtained through branch-PCR can carry large gene cargos and maintain a biocompatible nanoscale size, which we envisage will expand its medical applications in gene therapy.     

Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy

Precise editing of the genome of a living body is a goal pursued by scientists in many fields. In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome-editing systems have become a revolutionary toolbox for gene editing across various species. However, the low transfection efficiency of the CRISPR/Cas9 system to mammalian cells in vitro and in vivo is a big obstacle hindering wide and deep application. In this review, recently developed delivery strategies for various CRISPR/Cas9 formulations and their applications in treating gene-related diseases are briefly summarized. This review should inspire others to explore more efficient strategies for CRISPR system delivery and gene therapy.     

Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.     

Exosomes as Targeted Delivery Platform of CRISPR/Cas9 for Therapeutic Genome Editing

Therapeutic genome editing harnesses the power of genome editing tools to correct erroneous genes associated with disease pathology. To bring the CRISPR/Cas9 tool from the bench to the bedside, a critical hurdle is the safe and efficient delivery of this nucleic acid tool to the desired type of cells in patients. This review discusses the use of natural carriers, extracellular vesicles (EVs), in particular exosomes, to fill the gap. Exosomes are lipid-containing nanovesicle released by various types of cells to mediate cell-cell communications. Their inherent long-distance transportation capability, biocompatibility, and engineerability have made EVs potential vehicles for delivering therapeutic drugs. We summarize the recent progress of harnessing exosomes as delivery vehicles for the CRISPR/Cas system to achieve therapeutic gene editing for disease treatment, with a focus on various strategies to achieve selective delivery to a particular type of cell and efficient packaging of the genome editing tools in the vesicles. Critical issues and possible solutions in the design and engineering of the targeting vehicles are highlighted. Taken together, we demonstrate EV/exosome-mediated packaging of the nucleic acid/protein tools and the cell/tissue-targeted delivery to be a viable way towards the clinical translation of the CRISPR/Cas9 technology.     

First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.

Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.     

CRISPR/Cas9: Principle,Applications, and Delivery through Extracellular Vesicles

The establishment of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology for eukaryotic gene editing opened up new avenues not only for the analysis of gene function but also for therapeutic interventions. While the original methodology allowed for targeted gene disruption, recent technological advancements yielded a rich assortment of tools to modify genes and gene expression in various ways. Currently, clinical applications of this technology fell short of expectations mainly due to problems with the efficient and safe delivery of CRISPR/Cas9 components to living organisms. The targeted in vivo delivery of therapeutic nucleic acids and proteins remain technically challenging and further limitations emerge, for instance, by unwanted off-target effects, immune reactions, toxicity, or rapid degradation of the transfer vehicles. One approach that might overcome many of these limitations employs extracellular vesicles as intercellular delivery devices. In this review, we first introduce the CRISPR/Cas9 system and its latest advancements, outline major applications, and summarize the current state of the art technology using exosomes or microvesicles for transporting CRISPR/Cas9 constituents into eukaryotic cells.     

An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T₀ plants and 83.2% of T₁ plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T₁ progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.     

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 μM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.     

Targeting Cancer with CRISPR/Cas9-Based Therapy

Cancer is a devastating condition characterised by the uncontrolled division of cells with many forms remaining resistant to current treatment. A hallmark of cancer is the gradual accumulation of somatic mutations which drive tumorigenesis in cancerous cells, creating a mutation landscape distinctive to a cancer type, an individual patient or even a single tumour lesion. Gene editing with CRISPR/Cas9-based tools now enables the precise and permanent targeting of mutations and offers an opportunity to harness this technology to target oncogenic mutations. However, the development of safe and effective gene editing therapies for cancer relies on careful design to spare normal cells and avoid introducing other mutations. This article aims to describe recent advancements in cancer-selective treatments based on the CRISPR/Cas9 system, especially focusing on strategies for targeted delivery of the CRISPR/Cas9 machinery to affected cells, controlling Cas9 expression in tissues of interest and disrupting cancer-specific genes to result in selective death of malignant cells.