Editing System Research Articles

Restoration of Genetic Code in Macular Mouse Fibroblasts via APOBEC1-Mediated RNA Editing

RNA editing is a significant mechanism underlying genetic variation and protein molecule alteration; C-to-U RNA editing, specifically, is important in the regulation of mammalian genetic diversity. The ability to define and limit accesses of enzymatic machinery to avoid the modification of unintended targets is key to the success of RNA editing. Identification of the core component of the apoB RNA editing holoenzyme, APOBEC, and investigation into new candidate genes encoding other elements of the complex could reveal further details regarding APOBEC-mediated mRNA editing. Menkes disease is a recessive X-chromosome-linked hereditary syndrome in humans, caused by defective copper metabolism due to mutations in the ATP7A gene, which encodes a copper transport protein. Here, we generated plasmids encoding the MS2 system and the APOBEC1 deaminase domain and used a guide RNA with flanking MS2 sites to restore mutated Atp7a in fibroblasts from a macular mouse model of Menkes disease withs T>C mutation. Around 35% of the mutated C nucleotide (nt) was restored to U, demonstrating that our RNA editing system is reliable and has potential for therapeutic clinical application. RNA base editing via human RNA-guided cytidine deaminases is a potentially attractive approach for in vivo therapeutic application and provides opportunities for new developments in this field.

Macrophage-specific in vivo RNA editing promotes phagocytosis and antitumor immunity in mice.

Macrophages play a central role in antitumor immunity, making them an attractive target for gene therapy strategies. However, macrophages are difficult to transfect because of nucleic acid sensors that can trigger the degradation of foreign plasmid DNA. Here, we developed a macrophage-specific editing (MAGE) system by which compact plasmid DNA encoding a CasRx editor can be delivered to macrophages by a poly(β-amino ester) (PBAE) carrier to bypass the DNA sensor and enable RNA editing in vitro and in vivo. We identified a four-arm branched PBAE with 1-(2-aminoethyl)-4-methylpiperazine end-capping (PBAE29) that enables highly efficient macrophage transfection. PBAE29-mediated transfection of cultured macrophages stimulated less inflammatory cytokine production and inflammasome activation compared with traditional lipofectamine or electroporation-mediated plasmid delivery. Transfection efficiency was further improved by delivering CasRx by minicircle plasmid. The MAGE system incorporated a layer of carboxylated-mannan coating to target macrophage mannose receptors and a macrophage-specific promoter for enhanced selectivity. The delivery of CasRx with guide RNA targeting the transcripts for sialic acid-binding immunoglobulin similar to lectin 10 and signal regulatory protein alpha expression resulted in effective protein knockdown, improving macrophage phagocytosis. The MAGE system also showed efficacy in targeting macrophages in vivo, stimulating antitumor immune responses and reducing tumor volume in murine tumor models, including patient-derived pancreatic adenocarcinoma xenografts in humanized mice. In sum, the MAGE system presents a promising platform for in vivo macrophage-specific delivery of RNA editing tools that can be applied as a cancer therapy.

Mitochondrial DNA Structure in Trypanosoma cruzi

Kinetoplastids display a single, large mitochondrion per cell, with their mitochondrial DNA referred to as the kinetoplast. This kinetoplast is a network of concatenated circular molecules comprising a maxicircle (20–64 kb) and up to thousands of minicircles varying in size depending on the species (0.5–10 kb). In Trypanosoma cruzi, maxicircles contain typical mitochondrial genes found in other eukaryotes. They consist of coding and divergent/variable regions, complicating their assembly due to repetitive elements. However, next-generation sequencing (NGS) methods have resolved these issues, enabling the complete sequencing of maxicircles from different strains. Furthermore, several insertions and deletions in the maxicircle sequences have been identified among strains, affecting specific genes. Unique to kinetoplastids, minicircles play a crucial role in a particular U-insertion/deletion RNA editing system by encoding guide RNAs (gRNAs). These gRNAs are essential for editing and maturing maxicircle mRNAs. In Trypanosoma cruzi, although only a few studies have utilized NGS methods to date, the structure of these molecules suggests a classification into four main groups of minicircles. This classification is based on their size and the number of highly conserved regions (mHCRs) and hypervariable regions (mHVRs).

In vivo base editing extends lifespan of a humanized mouse model of prion disease.

Prion disease is a fatal neurodegenerative disease caused by the misfolding of prion protein (PrP) encoded by the PRNP gene. While there is currently no cure for the disease, depleting PrP in the brain is an established strategy to prevent or stall templated misfolding of PrP. Here we developed in vivo cytosine and adenine base strategies delivered by adeno-associated viruses to permanently modify the PRNP locus to achieve PrP knockdown in the mouse brain. Systemic injection of dual-adeno-associated virus PHP.eB encoding BE3.9max and single guide RNA installing PRNP R37X resulted in 37% average installation of the desired edit, 50% reduction of PrP in the mouse brain and 52% extension of lifespan in transgenic human PRNP mice inoculated with pathogenic human prion isolates representing the most common sporadic and genetic subtypes of prion disease. We further engineered base editing systems to achieve improved in vivo potency and reduced base editor expression in nontargeting tissues, resulting in 63% average PrP reduction in the mouse brain from a 6.7-fold lower viral dose, with no detected off-target editing of anticipated clinical significance observed in either human cells or mouse tissues. These findings support the potential of in vivo base editing as one-time treatment for prion disease.

Establishment of a CRISPR-Cas9-Mediated Genome Editing System in Flax.

Flax is an important crop used for oil and fiber production. Although genetic engineering has been possible in flax, it is not commonly used to produce cultivars. However, the use of genome editing technology, which can produce site-specific mutations without introducing foreign genes, may be a valuable tool for creating elite cultivars that can be easily cultivated. The purpose of this study was to investigate the potential of genome editing in flax by establishing the clustered regularly interspaced short palindromic repeats (CR ISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) genome editing system using the phytoene desaturase (PDS) gene, which produces albino mutants that are easily identifiable. Four sgRNAs were designed from two PDS genes of Flax (LuPDS1 and LuPDS2), and CRISPR-Cas9 genome editing vectors were constructed. After gene transformation, albino phenotypes were observed in transformed callus and regenerated plantlets on selection media. Polymerase chain reaction (PCR) amplification and sequencing of the PDS genes revealed deletions and insertions in the albino tissues, indicating successful editing of the PDS genes. Potential off-target sites were analyzed, but no off-target mutations were found, indicating the specificity of the CRISPR-Cas9 system. The establishment of a flax genome editing system using the CRISPR-Cas9 technology opens up new possibilities for the genetic engineering of flax. This study demonstrates the potential of genome editing in creating elite cultivars that can be easily cultivated, which can have significant implications for the flax industry.

Engineering an optimized hypercompact CRISPR/Cas12j-8 system for efficient genome editing in plants.

The Cas12j-8 nuclease, derived from the type V CRISPR system, is approximately half the size of Cas9 and recognizes a 5'-TTN-3' protospacer adjacent motif sequence, thus potentially having broad application in genome editing for crop improvement. However, its editing efficiency remains low in plants. In this study, we rationally engineered both the crRNA and the Cas12j-8 nuclease. The engineered crRNA and Cas12j-8 markedly improved genome editing efficiency in plants. When combined, they exhibited robust editing activity in soybean and rice, enabling the editing of target sites that were previously uneditable. Notably, for certain target sequences, the editing activity was comparable to that of SpCas9 when targeting identical sequences, and it outperformed the Cas12j-2 variant, nCas12j-2, across all tested targets. Additionally, we developed cytosine base editors based on the engineered crRNA and Cas12j-8, demonstrating an average increase of 5.36- to 6.85-fold in base-editing efficiency (C to T) compared with the unengineered system in plants, with no insertions or deletions (indels) observed. Collectively, these findings indicate that the engineered hypercompact CRISPR/Cas12j-8 system serves as an efficient tool for genome editing mediated by both nuclease cleavage and base editing in plants.

CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery.

To address a wide range of genetic diseases, genome editing tools that can achieve targeted delivery of large genes without causing double-strand breaks (DSBs) or requiring DNA templates are necessary. Here, we introduce CRISPR-Enabled Autonomous Transposable Element (CREATE), a genome editing system that combines the programmability and precision of CRISPR/Cas9 with the RNA-mediated gene insertion capabilities of the human LINE-1 (L1) element. CREATE employs a modified L1 mRNA to carry a payload gene, and a Cas9 nickase to facilitate targeted editing by L1-mediated reverse transcription and integration without relying on DSBs or DNA templates. Using this system, we demonstrate programmable insertion of a 1.1 kb gene expression cassette into specific genomic loci of human cell lines and primary T cells. Mechanistic studies reveal that CREATE editing is highly specific with no observed off-target events. Together, these findings establish CREATE as a programmable, RNA-based gene delivery technology with broad therapeutic potential.

Flexibility in PAM Recognition Expands DNA Targeting in xCas9.

xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.

CRISPR/Cas9-Mediated Gene-Knockout and In Situ Complementation System for Phytophthora sojae.

The establishment of reliable and efficient systems for genome editing in Phytophthora is very important for studying gene functions. Here, step-by-step methods for CRISPR/Cas9-based gene knockout and in situ complementation for Phytophthora sojae are presented. These steps include the sgRNA design, Cas9-sgRNA plasmid construction, homologous replacement, complementation vector construction, P. sojae transformation, and detection of mutations for both gene knockout and in situ complementation. These methods may also potentially be adapted for other Phytophthora species.

Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.

Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.

Efficient Genetic Transformation and Suicide Plasmid-mediated Genome Editing System for Non-model Microorganism Erwinia persicina.

Erwinia persicina is a gram-negative bacterium that causes diseases in plants. Recently, E. persicina BST187 was shown to exhibit broad-spectrum antibacterial activity due to its inhibitory effects on bacterial acetyl-CoA carboxylase, demonstrating promising potential as a biological control agent. However, the lack of suitable genetic manipulation techniques limits its exploitation and industrial application. Here, we developed an efficient transformation system for E. persicina. Using pET28a as the starting vector, the expression cassette of the red fluorescent protein-encoding gene with the strong promoter J23119 was constructed and transformed into BST187 competent cells to verify the overexpression system. Moreover, suicide plasmid-mediated genome editing systems was developed, and lacZ was knocked out of BST187 genome by parental conjugation transfer using the recombinant suicide vector pKNOCK-sacB-km-lacZ. Therefore, both the transformation and suicide plasmid-mediated genome editing system will greatly facilitate genetic manipulations in E. persicina and promote its development and application. Key features • Our studies establish a genetic manipulation system for Erwinia persicina, providing a versatile tool for studying the gene function of non-model microorganisms. • Requires approximately 6-10 days to complete modification of a chromosome locus.

Immunization with the NcMYR1 gene knockout strain effectively protected C57BL/6 mice and their pups against the Neospora caninum challenge

ABSTRACT Neospora caninum is an important protozoan parasite that causes abortion in cattle and nervous system dysfunction in dogs. No effective drugs and vaccines for neosporosis are available. Further elucidation of proteins related to N. caninum virulence will provide potential candidates for vaccine development against neosporosis. In the present study, N. caninum c-Myc regulatory protein (NcMYR1) gene knockout strains (ΔNcMYR1-1, ΔNcMYR1-2, and ΔNcMYR1-3) were generated using the CRISPR-Cas9 gene editing system to investigate phenotype changes and the potential of the ΔNcMYR1-1 strain as an attenuated vaccine, and this is the first time of using the N. caninum CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in N. caninum tachyzoites. The deficiency of NcMYR1 decreased the plaque area and the rate of invasion, replication, and egression of the parasites. ΔNcMYR1-1 strain-infected C57BL/6 mice had 100% survival rate, reduced parasite burden, and alleviated pathological changes in tissues compared with those in Nc-1 strain-infected mice. Immunization with ΔNcMYR1-1 tachyzoites increased the productions of cytokines in mice, with a survival rate reaching 80%, and the parasite burdens in the liver and spleen were greatly reduced when challenged with the Nc-1 strain with a lethal dose after 40 days of ΔNcMYR1-1 tachyzoite immunization. ΔNcMYR1 immunization could decrease the abortion rate of female mice from 71.4% to 12.5% and increase the survival rate of pups from 12.5% to 83.3% against the N. caninum challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against N. caninum infection and vertical transmission.

A single-plasmid-based, easily curable CRISPR/Cas9 system for rapid, iterative genome editing in Pseudomonas putida KT2440

BackgroundPseudomonas putida KT2440, a non-pathogenic soil bacterium, is a key platform strain in synthetic biology and industrial applications due to its robustness and metabolic versatility. Various systems have been developed for genome editing in P. putida, including transposon modules, integrative plasmids, recombineering systems, and CRISPR/Cas systems. However, rapid iterative genome editing is limited by complex and lengthy processes.ResultsWe discovered that the pBBR1MCS2 plasmid carrying the CRISPR/Cas9 module could be easily cured in P. putida KT2440 at 30 oC. We then developed an all-in-one CRISPR/Cas9 system for yqhD and ech-vdh-fcs deletions, respectively, and further optimized the editing efficiency by varying homology arm lengths and target sites. Sequential gene deletions of vdh and vanAB were carried out rapidly using single-round processing and easy plasmid curing. This system’s user-friendliness was validated by 3 researchers from two labs for 9 deletions, 3 substitutions, and 2 insertions. Finally, iterative genome editing was used to engineer P. putida for valencene biosynthesis, achieving a 10-fold increase in yield.ConclusionsWe developed and applied a rapid all-in-one plasmid CRISPR/Cas9 system for genome editing in P. putida. This system requires less than 1.5 days for one edit due to simplified plasmid construction, electroporation and curing processes, thus accelerating the cycle of genome editing. To our knowledge, this is the fastest iterative genome editing system for P. putida. Using this system, we rapidly engineered P. putida for valencene biosynthesis for the first time, showcasing the system’s potential for expanding biotechnological applications.

Development of an efficient and heritable virus-induced genome editing system in Solanum lycopersicum

Abstract The CRISPR-Cas9 system can be used to introduce site-specific mutations into the genome of tomato (Solanum lycopersicum) plants. However, the direct application of this revolutionary technology to desirable tomato cultivars has been hindered by the challenges of generating transgenic plants. To address this issue, we developed an efficient and heritable genome editing system using tobacco rattle virus (TRV) for an elite tomato cultivar (the paternal line of Saladette). Notably, this virus-induced genome editing (VIGE) system enables the rapid production of various mutant seeds without the need for additional plant transformation and tissue culture, once a Cas9-expressing tomato line is established. This VIGE system consists of transgenic tomato plants that express Cas9 under the control of the tomato ubiquitin 10 (SlUbi10) gene promoter and a mobile guide RNA scaffold (gRNA:SlmFT) generated using the sequence of the tomato Flowering Locus T (SlFT) gene. We determined its editing efficiency by targeting the tomato phytoene desaturase (SlPDS) gene, which causes photobleaching symptoms when disrupted. Most transgenic seedlings infected with the TRV vectors carrying the SlPDS-targeting sgRNA developed chimeric albino leaves associated with a high frequency of indel mutations in the SlPDS gene. Remarkably, fruits from these plants yielded homozygous SlPDS knockout seeds at rates ranging from 15% to 100%. These results demonstrate the exceptional effectiveness of our VIGE system in rapidly generating heritable genome edits in tomato.

Exploring Ethical Frontiers: A Content Analysis of Perceptions Surrounding Genetic Editing in Mental Health Treatments Over Time

Genetic editing has become a pivotal biotechnology tool in healthcare, enabling precise manipulation of DNA through insertions, deletions, modifications, or replacements. Its applications have expanded from treating monogenic diseases to addressing polygenic conditions, including potential mental health therapies. This study examines public sentiment and ethical considerations surrounding genetic editing systems like CRISPR- Cas9, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Analyzing a decade of medical literature, the research highlights a growing public acceptance of these technologies despite ethical concerns, such as off-target effects and unintended consequences. Positive sentiment trends indicate that social acceptance may support the integration of genetic editing in mental health treatments, driving advancements in healthcare. Addressing risks, including long-term impacts and ethical dilemmas, remains critical for responsible implementation. As public attitudes continue to improve, genetic editing may become a widely accepted therapeutic approach, fostering innovation in healthcare. Future research should focus on mitigating risks and enhancing the safety and efficacy of genetic editing to ensure its sustainable development and adoption.

Applications of CRISPR/Cas9 systems for gene editing in Bacillus spp.

Applications of CRISPR/Cas9 systems for gene editing in Bacillus spp.

Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a.

Single-cell lineage tracing based on CRISPR-Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool that utilizes two orthogonal gene editing systems to record cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability to enhance lineage recording with minimized target dropouts and potentially deeper tree depths. Applying DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers, showcased by identifying transcription factor Foxb1 as a modulator in the cell fate determination of neuromesodermal progenitors. Collectively, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.

DNA-free base editing in lettuce via in vitro transcribed base editors.

A newly developed RNA-based adenine and cytosine base editing system achieves targeted and efficient A-to-G and C-to-T conversions in lettuce. This DNA-free base editing method has potential uses in crop breeding and biotechnology.

A frameshift mutation in JAZ10 resolves the growth versus defense dilemma in rice

CRISPR-Cas9 genome editing systems have revolutionized plant gene functional studies by enabling the targeted introduction of insertion-deletions (INDELs) via the nonhomologous end-joining (NHEJ) pathway. Frameshift-inducing INDELs can introduce a premature termination codon and, in other instances, can lead to the appearance of new proteins. Here, we found that mutations in the rice jasmonate (JA) signaling gene OsJAZ10 by CRISPR-Cas9-based genome editing did not affect canonical JA signaling. However, a type of mutant with an INDEL that yielded a novel frameshift protein named FJ10 (Frameshift mutation of JAZ10), exhibited enhanced rice growth and increased resistance to brown planthopper attacks. Overexpression of FJ10 in wild-type plants phenocopies OsJAZ10 frameshift mutants. Further characterization revealed that FJ10 interacts with Slender Rice 1 (OsSLR1) and F-box/Kelch 16 (OsFBK16). These interactions disrupt the function of OsSLR1 in suppressing gibberellin-mediated growth and the function of OsFBK16 in repressing lignin-mediated defense responses, respectively. Field experiments with FJ10-expressing plants demonstrate that this protein uncouples the growth-defense tradeoff, opening broad avenues to obtain cultivars with enhanced yield without compromised defenses.

Bidirectional Prime Editing: Combining Precision with Versatility for Genome Editing.

Genome editing techniques have potential to revolutionize the field of life sciences. Several limitations associated with traditional gene editing techniques have been resolved with the development of prime editors that precisely edit the DNA without double-strand breaks (DSBs). To further improve the efficiency, several modified versions of prime editing (PE) system have been introduced. Bi-directional PE (Bi-PE), for example, uses two PE guide RNAs enabling broad and improved editing efficiency. It has the potential to alter, delete, integrate, and replace larger genome sequences and edit multiple bases at the same time. This review aims to discuss the typical gene editing methods that offer DSB-mediated repair mechanisms, followed by the latest advances in genome editing technologies with non-DSB-mediated repair. The review specifically focuses on Bi-PE being an efficient tool to edit the human genome. In addition, the review discusses the applications, limitations, and future perspectives of Bi-PE for gene editing.