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CRISPR-PN2: a flexible and genome-aware platform for diverse CRISPR experiments in parasitic nematodes.
Parasitic nematodes represent a significant threat to human health, causing diseases of major socioeconomic importance worldwide. Central to controlling infections of parasitic nematodes is a more detailed molecular picture of host specificity, parasite activation and immune suppression. CRISPR technology holds huge potential for researchers in the field of parasitic nematology, as it provides a powerful genetic tool to dissect questions in parasite biology. To expedite the development of CRISPR technology in parasitic nematodes, software is required to facilitate the design of effective and specific sgRNA sequences. Here, the author introduces CRISPR-PN2, a comprehensive web-based platform that provides flexible use control over the automated design of specific gRNA sequences for CRISPR experiments in parasitic nematodes.
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"Doing CRISPR".
Salmon farming is a key industry in Norway, with firsthand value of more than 60 billion Norwegian crowns in 2017. The salmon industry is a driving force for biotechnological applications in the marine sector. The recent release of the Atlantic salmon reference genome offers new opportunities to solve major aquaculture bottlenecks that currently limit expansion of the industry. One major bottleneck is the genetic impact of escaped farmed salmon on wild populations. To solve this problem, the industry can use sterile salmon in production. As shown by Wargelius et al., sterile salmon can be made by preventing the formation of germ cells through genome editing using the CRISPR-Cas9 method. This approach solves problems of genetic introgression and precocious maturation. However, genome editing of animals, especially for human consumption, raises ethical as well as safety and legal questions. These social and ethical aspects can have tremendous impact in analyzing the final result of salmon farming (e.g., consumer acceptability of a fresh or frozen filet or similar salmon product) but also can be examined "upstream" by describing and assessing the research communities that promote and carry out the science that underpins the salmon industry. Who produces the scientific "facts" that govern the Norwegian aquaculture industry? How do these scientific communities work together? What are the societal impacts of this science? This article uses ethnographical observation and interviews to describe the state-of-the-art of CRISPR gene-editing procedures currently employed in the science and industry collaboration in Norway.
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Generating Genetically Modified Mice: A Decision Guide.
The generation of a new genetically modified mouse strain is a big hurdle to take for many researchers. It is often unclear which steps and decisions have to be made prior to obtaining the desired mouse model. This review aims to help researchers by providing a decision guide that answers the essential questions that need to be asked before generating the most suitable genetically modified mouse line in the most optimal timeframe. The review includes the latest technologies in both the stem cell culture and gene editing tools, particularly CRISPR/Cas9, and provides compatibility guidelines for selecting among the different types of genetic modifications that can be introduced in the mouse genome and the various routes for introducing these modifications into the mouse germline.
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Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases.
Directed evolution and targeted genome editing have been deployed to create genetic variants with usefully altered phenotypes. However, these methods are limited to high-throughput screening methods or serial manipulation of single genes. In this study, we implemented multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) to simultaneously target up to 11 sites on the Escherichia coli chromosome for multiplex gene interruption and/or insertion, generating combinatorial genomic diversity. The MUCICAT system was improved by replacing the isopropyl-beta-D-thiogalactoside (IPTG)-dependent promoter to decouple gene editing and product synthesis and truncating the right end to reduce the leakage expression of cargo. We applied MUCICAT to engineer and optimize the N-acetylglucosamine (GlcNAc) biosynthesis pathway in E. coli to overproduce the industrially important GlcNAc in only 8 days. Two rounds of transformation, the first round for disruption of two degradation pathways related gene clusters and the second round for multiplex integration of the GlcNAc gene cassette, would generate a library with 1-11 copies of the GlcNAc cassette. We isolated a best variant with five copies of GlcNAc cassettes, producing 11.59 g/L GlcNAc, which was more than sixfold than that of the strain containing the pET-GNAc plasmid. Our multiplex approach MUCICAT has potential to become a powerful tool of cell programing and can be widely applied in many fields such as synthetic biology.
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A comparison between CARLIN and DNA Typewriter in CRISPR-mediated lineage tracing.
BACKGROUND: CARLIN and DNA Typewriter are two major breakthroughs in CRISPR-based lineage tracing technology. It is essential to understand the potential and performance of these methods in lineage tracing, which provides important guidance on experimental design. RESULTS: In this study, we systematically compare these two strategies using a unified stochastic simulation framework with known ground-truth lineages. By explicitly modeling CRISPR editing dynamics, barcode evolution, and cell division processes, the framework enables quantitative benchmarking of lineage reconstruction accuracy across diverse experimental parameter regimes. Both methods are evaluated using multiple accuracy metrics, including Robinson-Foulds accuracy and triplet accuracy, allowing a comprehensive assessment of lineage reconstruction performance under various editing probabilities, sampling depths, and lineage lengths. CONCLUSIONS: DNA Typewriter consistently outperforms CARLIN in lineage reconstruction accuracy when sufficient numbers of recording targets are used, particularly in more cell divisions. Sequential and ordered recording in DNA Typewriter substantially reduces ambiguity in lineage inference compared to unordered CRISPR barcode editing. CARLIN's lineage-recording potential exhausts rapidly under continuous induction, limiting its effectiveness in long-term lineage tracing. Triplet accuracy provides a more permissive and informative metric than Robinson-Foulds accuracy, especially under partial sampling scenarios.
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Prime Editing Guide RNA Design Automation Using PINE-CONE.
CRISPR-based technologies are paramount in genome engineering and synthetic biology. Prime editing (PE) is a technology capable of installing genomic edits without double-stranded DNA breaks (DSBs) or donor DNA. Prime editing guide RNAs (pegRNAs) simultaneously encode both guide and edit template sequences. They are more design intensive than CRISPR single guide RNAs (sgRNAs). As such, application of PE technology is hindered by the limited throughput of manual pegRNA design. To that end, we designed a software tool, Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE), that enables high-throughput automated design of pegRNAs and prime editing strategies. PINE-CONE translates edit coordinates and sequences into pegRNA designs, accessory guides, and oligonucleotides for facile cloning workflows. To demonstrate PINE-CONE's utility in studying disease-relevant genotypes, we rapidly design a library of pegRNAs targeting Alzheimer's Disease single nucleotide polymorphisms (SNPs). Overall, PINE-CONE will accelerate the application of PEs in synthetic biology and biomedical research.
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Development and application of a fast and efficient CRISPR/Cas12f -based genetic toolkit in Bacillus cereus GW-01.
Bacillus cereus GW-01, an efficient degrader of β-cypermethrin (β-CY), has a high safety profile and probiotic potential for regulating intestinal flora and fermented foods, which is difficult to genetically engineer for modification due to its restrictive modification system. This study successfully developed a CRISPR/enCas12f-based genome editing system, first selecting the plcR gene for proof-of-concept validation with 100% knockout efficiency. Subsequently, this system was utilized to delete the virulence gene nheABC in GW-01, yielding a safer probiotic strain. Compared with the wild-type strain GW-01, the probiotic-related indicators of the ΔnheABC mutant, including cell surface hydrophobicity, auto-aggregation ability and biofilm formation ability, were 80%, 90% and 2.9 (OD₅₉₅), respectively. There were no significant differences in these indicators between the mutant and the wild type. Meanwhile, the ΔnheABC mutant still maintained a high β-cypermethrin degradation efficiency of 80% at the concentration of 30 μg/mL. This work facilitates functional genomic research and genetic modification of Bacillus cereus GW-01. The established CRISPR/enCas12f system enables targeted gene deletion to explore gene functions and phenotypic mechanisms, and paves the way for its development into safe probiotics and excellent microbial chassis.
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Rational engineering of a modular bacterial CRISPR-Cas activation platform with expanded target range.
CRISPR-Cas activator (CRISPRa) systems that selectively turn on transcription of a target gene are a potentially transformative technology for programming cellular function. While in eukaryotes versatile CRISPRa systems exist, in bacteria these systems suffer from a limited ability to activate different genes due to strict distance-dependent requirements of functional target binding sites, and require greater customization to optimize performance in different genetic and cellular contexts. To address this, we apply a rational protein engineering approach to create a new CRISPRa platform that is highly modular to allow for easy customization and has increased targeting flexibility through harnessing engineered Cas proteins. We first demonstrate that transcription activation domains can be recruited by CRISPR-Cas through noncovalent protein-protein interactions, which allows each component to be encoded on separate and easily interchangeable plasmid elements. We then exploit this modularity to rapidly screen a library of different activation domains, creating new systems with distinct regulatory properties. Furthermore, we demonstrate that by harnessing a library of circularly permuted Cas proteins, we can create CRISPRa systems that have different target binding site requirements, which together, allow for expanded target range.
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CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.
In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.
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Establishment of a Cleavage-Based Single-Plasmid Dual-Luciferase Surrogate Reporter for the Cleavage Efficiency Evaluation of CRISPR-Cas12a Systems and Its Primary Application.
CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing.
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