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Optimized metrics for orthogonal combinatorial CRISPR screens.
CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.
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Transformed bovine trophoblast stem cell lines, characterization, gene editing and secretion†.
Trophoblast stem cells (TSCs) serve as a critical model for understanding placental development, early embryo-maternal interactions, and pregnancy establishment in mammals. In cattle, the developing trophectoderm plays an essential role in conceptus elongation and secretion of factors necessary for maternal recognition of pregnancy. Building on previous work identifying signaling pathways regulating bovine TSC self-renewal and differentiation, we report the generation and characterization of transformed bovine TSC (bTSC) lines derived from blastocysts via lentiviral transduction of simian vacuolating virus 40 large T antigen. These rapidly proliferating TSC cell lines, maintained in the presence of Rho-associated protein kinase (ROCK) inhibition, retain key morphological and transcriptional characteristics of bovine TSCs. Upon transforming growth factor β-induced differentiation, they exhibit morphological and molecular changes consistent with trophoblast maturation. To evaluate their utility for functional studies, we demonstrated stable gene introduction of tdTomato and EGFP using lentiviral vectors and employed CRISPR/Cas9-mediated gene editing to target lentiviral EGFP integration sites, confirming efficient gene deletion. Additionally, proteomic analysis of conditioned medium identified secreted proteins with potential roles in embryo-uterine interactions, aligning with factors previously reported in bovine conceptus secretomes. These findings establish transformed bTSC lines as a valuable model for investigating bovine trophoblast biology, functional gene studies, and trophoblast-endometrial signaling. By providing a renewable in vitro system with stable proliferative capacity, these cell lines enable further exploration of the molecular mechanisms governing early pregnancy in cattle.
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Novel approaches for HTLV-1 therapy: innovative applications of CRISPR-Cas9.
The human T-cell lymphotropic virus type 1 (HTLV-1) is a single-stranded positive-sense RNA virus that belongs to the Retroviridae family, genus Deltaretro, and infects approximately five to 10 million people worldwide. Although a significant number of individuals living with HTLV-1 remain asymptomatic throughout their lives, some develop one or more severe clinical conditions, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive and debilitating disease, and/or a subtype of non-Hodgkin's lymphoma with a more threatening course known as adult T-cell leukemia/lymphoma (ATLL). Moreover, current therapeutic options are limited and focus primarily on treating symptoms and controlling viral latency. CRISPR-Cas9 gene editing is proposed as a promising tool to address the intricate links associated with HTLV-1. By targeting or silencing key genes during initial infection and dysregulating immune signaling pathways, CRISPR-Cas9 offers potential intervention opportunities. In this review, we address the therapeutic potential of CRISPR-Cas9 gene editing, as well as examine the primary mechanisms involved in editing potential target genes and discuss the existing evidence in the current scientific literature.
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Synthetic chimeric nucleases function for efficient genome editing.
CRISPR-Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demonstration opens up the possibility of generating nuclease sequences with implications across biotechnology.
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Genome editing for primary immunodeficiencies: A therapeutic perspective on Wiskott-Aldrich syndrome.
Primary immunodeficiency diseases (PIDs) are a group of rare inherited disorders affecting the immune system that can be conventionally treated with allogeneic hematopoietic stem cell transplantation and with experimental autologous gene therapy. With both approaches still facing important challenges, gene editing has recently emerged as a potential valuable alternative for the treatment of genetic disorders and within a relatively short period from its initial development, has already entered some landmark clinical trials aimed at tackling several life-threatening diseases. In this review, we discuss the progress made towards the development of gene editing-based therapeutic strategies for PIDs with a special focus on Wiskott - Aldrich syndrome and outline their main challenges as well as future directions with respect to already established treatments.
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Trans-nuclei CRISPR/Cas9: safe approach for genome editing in the edible mushroom excluding foreign DNA sequences.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted genome editing has been applied to several major edible agaricomycetes, enabling efficient gene targeting. This method is promising for rapid and efficient breeding to isolate high-value cultivars and overcome cultivation challenges. However, the integration of foreign DNA fragments during this process raises concerns regarding genetically modified organisms (GMOs) and their regulatory restrictions. In this study, we developed a foreign-DNA-free genome editing method in Pleurotus ostreatus by transferring the Cas9/guide RNA (gRNA) complex between nuclei in the dikaryotic state. We isolated a donor monokaryotic P. ostreatus strain expressing Cas9 and gRNA targeting pyrG by introducing a recombinant plasmid, which exhibited uracil auxotrophy and 5-fluoroorotic acid (5-FOA) resistance. This strain was then crossed with a pyrG+ recipient monokaryon, resulting in dikaryotic strains exhibiting 5-FOA resistance after mycelial growth. When these strains were de-dikaryonized into monokaryons through protoplasting, we obtained monokaryotic isolates harboring the recipient nucleus with small indels at the pyrG target site. Importantly, these isolates were confirmed to be free of foreign DNA through genomic PCR, Southern blotting, and whole-genome resequencing analyses. This is the first report of an efficient genome editing protocol in agaricomycetes that ensures no integration of exogenous DNA. This approach is expected to be applicable to other fungi with a dikaryotic life cycle, opening new possibilities for molecular breeding without the concerns associated with GMOs. KEY POINTS: • Successful genome editing via CRISPR/Cas9 trans-nuclei manner in P. ostreatus. • Recipient monokaryons from gene-edited dikaryons showed no exogenous DNA sequences. • Efficient genome editing protocol for safer molecular breeding in mushroom fungus.
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Co-Delivery of Multiple RNAs via Lipid Nanoparticles Enables Precise Gene Editing of CAR-T Cells.
Over the past decade, CAR-T cell therapy has achieved remarkable success in treating hematological malignancies. However, traditional CAR-T cell engineering employs viral vectors, which has several limitations. Additionally, the immunosuppressive tumor microenvironment, particularly mediated by the PD-1/PD-L1 pathway, significantly restricts CAR-T cell efficacy. CRISPR/Cas9-mediated PD-1 knockout can enhance CAR-T cell anti-tumor activity, but traditional electroporation (EP) method often damages T cells. Herein, a novel lipid nanoparticles (LNPs)-mediated delivery technology are introduced to engineer CAR-T cells. The LNPs platform enables the simultaneous expression of CAR cassette and CRISPR/Cas9 gene editor in T cells via co-delivery of multiplex RNAs (CD19 CAR mRNA+Cas9 mRNA+sgRNA targeting PD-1). Importantly, LNPs exhibit higher transfection efficiency and superior cell viability compared to traditional electroporation method. The engineered CAR-T cells with PD-1 knockout, which express anti-CD19 CAR, can specifically kill CD19+ Nalm-6 tumor cells in vitro and display enhanced anti-tumor activity in vivo. Furthermore, LNPs-mediated co-delivery of Cas9 mRNA and sgRNAs targeting PD-1, TRAC, and B2M enables triple-knockout of T cells with high editing efficiencies (76% for PD-1, 86% for TRAC, and 80% for B2M), highlighting the ability for multiplex gene editing. This LNP-mediated delivery strategy has great potentials for the development of safer and more efficacious CAR-T cells.
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Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat.
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The investigation of the mechanism underlying variations in oxidative stress tolerance of Lacticaseibacillus paracasei resulting from fermentation methods through endogenous CRISPR-Cas9 editing methodology.
The probiotic effects of lactic acid bacteria make them widely used in human and animal breeding industry. However, the presence of oxidative stress during the production and application process can cause bacterial damage or even death, significantly compromising the functionality of probiotics. Despite its potential for broader application scenarios that could provide a more comprehensive understanding of bacteria's internal adaptation strategies, there is a lack of research investigating oxidative stress from the perspective of culture methods. In this study, the tolerance to oxidative stress was compared between bacteria cultivated through solid-state fermentation (SSF) and liquid-state fermentation (LSF), and the physiological and transcriptional disparities between these two bacterial strains were investigated. Additionally, a novel and efficient gene editing method was developed to elucidate the genetic basis underlying these differences in tolerance. The results demonstrated a significantly higher tolerance to oxidative stress in SSF bacteria compared to LSF bacteria, along with a stronger capacity for maintaining intracellular microenvironment stability and the activity of key metabolic enzymes. It is noteworthy that the bacteria from SSF significantly enhance the transport of carbohydrate substances and facilitate intracellular metabolic flow. Gene editing experiments have confirmed the crucial role of genes glpF and glpO in regulating the glycerol metabolism pathway, which is essential for enhancing the tolerance of bacteria from SSF to oxidative stress. Based on these findings, the mechanism underlying the disparity in oxidative stress tolerance resulting from different culture methods has been summarized. Furthermore, investigation into different culture modes has revealed that moderate oxygen levels during cultivation significantly influence variation in bacterial tolerance to oxidative stress. Importantly, these variations are species-specific and depend on the ecological niche distribution of Lactobacilli. These findings elucidate a novel mechanism by which Lacticaseibacillus paracasei Zhang tolerates oxidative stress, and also suggest that distinct cultivation and processing methods should be tailored based on the specific Lactobacilli groups to achieve optimal application effects in production.
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Standardized Iterative Genome Editing Method for Escherichia coli Based on CRISPR-Cas9.
The introduction of complex biosynthetic pathways into the hosts' chromosomes is gaining attention with the development of synthetic biology. While CRISPR-Cas9 has been widely employed for gene knock-in, the process of multigene insertion remains cumbersome due to laborious and empirical gene cloning procedures. To address this, we devised a standardized iterative genome editing system for Escherichia coli, harnessing the power of CRISPR-Cas9 and MetClo assembly. This comprehensive toolkit comprises two fundamental elements based on the Golden Gate standard for modular assembly of sgRNA or CRISPR arrays and donor DNAs. We achieved a gene insertion efficiency of up to 100%, targeting a single locus. Expression of tracrRNA using a strong promoter enhances multiplex genomic insertion efficiency to 7.3%, compared with 0.76% when a native promoter is used. To demonstrate the robust capabilities of this genome editing toolbox, we successfully integrated 5-10 genes from the coenzyme B12 biosynthetic pathway ranging from 5.3 to 8 Kb in length into the chromosome of E. coli chassis cells, resulting in 14 antibiotic-free, plasmid-free producers. Following an extensive screening process involving genes from diverse sources, cistronic design modifications, and chromosome repositioning, we obtained a recombinant strain yielding 1.49 mg L-1 coenzyme B12, the highest known titer achieved by using E. coli as the producer. Illuminating its user-friendliness, this genome editing system is an exceedingly versatile tool for expediently integrating complex biosynthetic pathway genes into hosts' genomes, thus facilitating pathway optimization for chemical production.
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