CRISPR-Mediated Viral Gene Knockout to Investigate Viral Evasion of Antiviral Innate Immunity.

The innate immune system relies on a variety of pathogen recognition receptors (PRRs) as the first line of defense against pathogenic invasions. Viruses have evolved multiple strategies to evade the host immune system through coevolution with hosts. The CRISPR-Cas system is an adaptive immune system in bacteria or archaea that defends against viral reinvasion by targeting nucleic acids for cleavage. Based on the characteristics of Cas proteins and their variants, the CRISPR-Cas system has been developed into a versatile gene-editing tool capable of gene knockout or knock-in operations to achieve genetic variations in organisms. It is now widely used in the study of viral immune evasion mechanisms. This chapter will introduce the use of the CRISPR-Cas9 system for editing herpes simplex virus 1 (HSV-1) genes to explore the mechanisms by which HSV-1 evades host innate immunity and the experimental procedures involved.

CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.

With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.

Modular DNA Construct Design for High-Throughput Golden Gate Assembly.

Golden Gate cloning enables the modular assembly of DNA parts into desired synthetic genetic constructs. The "one-pot" nature of Golden Gate reactions makes them particularly amenable to high-throughput automation, facilitating the generation of thousands of constructs in a massively parallel manner. One potential bottleneck in this process is the design of these constructs. There are multiple parameters that must be considered during the design of an assembly process, and the final design should also be checked and verified before implementation. Doing this by hand for large numbers of constructs is neither practical nor feasible and increases the likelihood of introducing potentially costly errors. In this chapter we describe a design workflow that utilizes bespoke computational tools to automate the key phases of the construct design process and perform sequence editing in batches.

Combining Oligo Pools and Golden Gate Cloning to Create Protein Variant Libraries or Guide RNA Libraries for CRISPR Applications.

Oligo pools are array-synthesized, user-defined mixtures of single-stranded oligonucleotides that can be used as a source of synthetic DNA for library cloning. While currently offering the most affordable source of synthetic DNA, oligo pools also come with limitations such as a maximum synthesis length (approximately 350 bases), a higher error rate compared to alternative synthesis methods, and the presence of truncated molecules in the pool due to incomplete synthesis. Here, we provide users with a comprehensive protocol that details how oligo pools can be used in combination with Golden Gate cloning to create user-defined protein mutant libraries, as well as single-guide RNA libraries for CRISPR applications. Our methods are optimized to work within the Yeast Toolkit Golden Gate scheme, but are in principle compatible with any other Golden Gate-based modular cloning toolkit and extendable to other restriction enzyme-based cloning methods beyond Golden Gate. Our methods yield high-quality, affordable, in-house variant libraries.

Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis.

Phage engineering is an emerging technology due to the promising potential application of phages in medical and biotechnological settings. Targeted phage mutagenesis tools are required to customize the phages for a specific application and generate, in addition to that, so-called designer phages. CRISPR-Cas technique is used in various organisms to perform targeted mutagenesis. Yet, its efficacy is notably limited for phage mutagenesis due to the highly abundant phage DNA modifications. Addressing this challenge, we have developed a novel approach that involves the temporal removal of phage DNA cytosine modifications, allowing for effective CRISPR-Cas targeting and subsequent introduction of mutations into the phage genome. The removal of cytosine modification relies on the catalytic activity of a eukaryotic ten-eleven translocation methylcytosine (TET) dioxygenase. TET enzymes iteratively de-modify methylated or hydroxymethylated cytosines on phage DNA. The temporal removal of cytosine modification ultimately enables efficient DNA cleavage by Cas enzymes and facilitates mutagenesis. To streamline the application of the coupled TET-CRISPR-Cas system, we use Golden Gate cloning for fast and efficient assembly of a vector that comprises a TET oxidase and a donor DNA required for scarless site-specific phage mutagenesis. Our approach significantly advances the engineering of modified phage genomes, enabling the efficient generation of customized phages for specific applications.

Protocol for NT-CRISPR: A Method for Efficient Genome Engineering in Vibrio natriegens.

Vibrio natriegens is a gram-negative bacterium, which has received increasing attention due to its very fast growth with a doubling time of under 10 min under optimal conditions. To enable a wide range of projects spanning from basic research to biotechnological applications, we developed NT-CRISPR as a new method for genome engineering. This book chapter provides a step-by-step protocol for the use of this previously published tool. NT-CRISPR combines natural transformation with counterselection through CRISPR-Cas9. Thereby, genomic regions can be deleted, foreign sequences can be integrated, and point mutations can be introduced. Furthermore, up to three simultaneous modifications are possible.

CRISPR is easy: Exposure to Last Week Tonight enhances knowledge about gene editing.

Experts have called for public engagement with the governance of controversial scientific research and discoveries, including CRISPR, the technology that enables gene editing. Though engaging and informing citizens who are not interested in the issue is a challenge, recent studies suggest humor has potential to close interest and knowledge gaps. We tested this potential by exposing individuals (N = 303) to one of three videos (an edited clip from Last Week Tonight, an edited clip from 60 Minutes, or control) that contained broadly overlapping facts about gene editing in an online survey. Results show that while exposure to the Last Week Tonight clip did not increase attentiveness to the issue of human gene editing among individuals with lower levels of interest in science, exposure to the humorous clip caused a modest improvement in issue knowledge. Positive main effects on perceived knowledge were found for both treatments. More research is needed but findings suggest that the use of humor in science communication offers potential, though perhaps limited, for broadening public engagement with emerging areas of science.

Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model.

BACKGROUND: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. METHODS: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status. RESULTS: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. CONCLUSIONS: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.

Versatile filamentous fungal host highly-producing heterologous natural products developed by genome editing-mediated engineering of multiple metabolic pathways.

Natural secondary metabolites are medically, agriculturally, and industrially beneficial to humans. For mass production, a heterologous production system is required, and various metabolic engineering trials have been reported in Escherichia coli and Saccharomyces cerevisiae to increase their production levels. Recently, filamentous fungi, especially Aspergillus oryzae, have been expected to be excellent hosts for the heterologous production of natural products; however, large-scale metabolic engineering has hardly been reported. Here, we elucidated candidate metabolic pathways to be modified for increased model terpene production by RNA-seq and metabolome analyses in A. oryzae and selected pathways such as ethanol fermentation, cytosolic acetyl-CoA production from citrate, and the mevalonate pathway. We performed metabolic modifications targeting these pathways using CRISPR/Cas9 genome editing and demonstrated their effectiveness in heterologous terpene production. Finally, a strain containing 13 metabolic modifications was generated, which showed enhanced heterologous production of pleuromutilin (8.5-fold), aphidicolin (65.6-fold), and ophiobolin C (28.5-fold) compared to the unmodified A. oryzae strain. Therefore, the strain generated by engineering multiple metabolic pathways can be employed as a versatile highly-producing host for a wide variety of terpenes.

Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes.

Deciphering the context-specific relationship between sequence and function is a major challenge in genomics. Existing tools for inducing locus-specific hypermutation and evolution in the native genome context are limited. Here we present a programmable platform for long-range, locus-specific hypermutation called helicase-assisted continuous editing (HACE). HACE leverages CRISPR-Cas9 to target a processive helicase-deaminase fusion that incurs mutations across large (>1000-base pair) genomic intervals. We applied HACE to identify mutations in mitogen-activated protein kinase kinase 1 (MEK1) that confer kinase inhibitor resistance, to dissect the impact of individual variants in splicing factor 3B subunit 1 (SF3B1)-dependent missplicing, and to evaluate noncoding variants in a stimulation-dependent immune enhancer of CD69. HACE provides a powerful tool for investigating coding and noncoding variants, uncovering combinatorial sequence-to-function relationships, and evolving new biological functions.

Expression and purification of cell-penetrating Cas9 and Cas12a enzymes for peptide-assisted genome editing.

Recent advances in CRISPR-Cas genomic editors have shifted us ever closer to achieving the ultimate therapeutic goal of accomplishing any edit in any cell. However, delivery of this editing machinery to primary cells with high efficiency while avoiding cellular toxicity remains a formidable challenge. Peptide-Assisted Genome Editing (PAGE) provides a simple, modular, and rapid approach for the protein-based delivery of CRISPR-Cas proteins or ribonucleoprotein complexes into primary cells with high efficiency and minimal cytotoxicity. In this chapter, we detail an expression and purification protocol to obtain highly pure Cas9-T6N and opCas12a-T8N PAGE genomic editors. The robustness of this protocol allows for consistent preparations of the purified editors that can be reliably used for the editing of primary and immortalized cells.

CRISPR-Cas9 guided RNA-based model for the silencing of spinal bulbar muscular atrophy: A functional genetic disorder.

This study explores a novel therapeutic approach for spinal bulbar muscular atrophy (SBMA), a neurodegenerative disorder caused by a mutation in the Androgen Receptor (AR) gene. The aim is to investigate the potential of CRISPR-Cas9 technology in targeting the mutant AR gene to inhibit its production. The objectives include assessing the accuracy and efficacy of CRISPR-Cas9 guided RNAs in silencing the mutant gene and evaluating the feasibility of this approach as a treatment for SBMA. Computational and in-silico approaches are used to evaluate the feasibility of using CRISPR-Cas9 technology for treating SBMA. Computational analysis is used to design CRISPR-Cas9 guided RNAs targeting the mutant AR gene, assessing their on-target and off-target scores, GC content, and structural accuracy. In-silico simulations predict the potential therapeutic outcomes of the CRISPR-Cas9 approach in an artificial environment. Three guided RNA (gRNA) sequences were designed using the CHOPCHOP tool, targeting specific regions of the AR gene with high efficiency and 100% match. These gRNAs demonstrated effective targeting with minimal off-target scores and optimal GC content. Additionally, lentiCRISPR v2 plasmids were designed for the delivery of CRISPR materials, enabling high-efficiency multiplex genome editing of the AR gene. Thermodynamic ensemble predictions indicated favorable secondary structure stability of the designed gRNAs, further supporting their suitability for gene editing. The evaluation of designed gRNAs confirmed their strong binding ability to the target sequences, validating their potential as effective tools for genome editing. The study highlights the potential of CRISPR-Cas9 technology for targeting the Androgen Receptor gene associated with spinal bulbar muscular atrophy (SBMA). The findings support the feasibility of this approach for gene editing and suggest further exploration in preclinical and clinical settings. Recommendations include continued research to optimize CRISPR-Cas9 delivery methods and enhance specificity for therapeutic applications in SBMA.

Toward a CRISPR-based mouse model of Vhl-deficient clear cell kidney cancer: Initial experience and lessons learned.

CRISPR is revolutionizing the ability to do somatic gene editing in mice for the purpose of creating new cancer models. Inactivation of the VHL tumor suppressor gene is the signature initiating event in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). Such tumors are usually driven by the excessive HIF2 activity that arises when the VHL gene product, pVHL, is defective. Given the pressing need for a robust immunocompetent mouse model of human ccRCC, we directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL and other known/suspected ccRCC tumor suppressor genes into the kidneys of C57BL/6 mice under conditions where Cas9 was under the control of one of two different kidney-specific promoters (Cdh16 or Pax8) to induce kidney tumors. An AAV targeting Vhl, Pbrm1, Keap1, and Tsc1 reproducibly caused macroscopic ccRCCs that partially resembled human ccRCC tumors with respect to transcriptome and cell of origin and responded to a ccRCC standard-of-care agent, axitinib. Unfortunately, these tumors, like those produced by earlier genetically engineered mouse ccRCCs, are HIF2 independent.

Genetic manipulation of the genes for clonal seeds results in sterility in cotton.

BACKGROUND: Heterosis is a common phenomenon in plants and has been extensively applied in crop breeding. However, the superior traits in the hybrids can only be maintained in the first generation but segregate in the following generations. Maintaining heterosis in generations has been challenging but highly desirable in crop breeding. Recent study showed that maternally produced diploid seeds could be achieved in rice by knocking out three meiosis related genes, namely REC8, PAIR1, OSD1 to create MiMe in combination with egg cell specific expression of BBM transcription factor, a technology called clonal seeds. Interestingly, there has been very limited reports indicating the feasibility of this approach in other crops. RESULTS: In this study, we aimed to test whether clonal seeds could be created in cotton. We identified the homologs of the three meiosis related genes in cotton and used the multiplex CRISPR/Cas9 gene editing system to simultaneously knock out these three genes in both A and D sub-genomes. More than 50 transgenic cotton plants were generated, and fragment analysis indicated that multiple gene knockouts occurred in the transgenic plants. However, all the transgenic plants were sterile apparently due to the lack of pollen. Pollination of the flowers of the transgenic plants using the wild type pollens could not generate seeds, an indication of defects in the formation of female sexual cells in the transgenic plants. In addition, we generated transgenic cotton plants expressing the cotton BBM gene driven by the Arabidopsis egg cell specific promoter pDD45. Two transgenic plants were obtained, and both showed severely reduced fertility. CONCLUSIONS: Overall, our results indicate that knockout of the clonal seeds related genes in cotton causes sterility and how to manipulate genes to create clonal seeds in cotton requires further research.

CRISPR-edited human ES-derived oligodendrocyte progenitor cells improve remyelination in rodents.

In Multiple Sclerosis (MS), inflammatory demyelinated lesions in the brain and spinal cord lead to neurodegeneration and progressive disability. Remyelination can restore fast saltatory conduction and neuroprotection but is inefficient in MS especially with increasing age, and is not yet treatable with therapies. Intrinsic and extrinsic inhibition of oligodendrocyte progenitor cell (OPC) function contributes to remyelination failure, and we hypothesised that the transplantation of 'improved' OPCs, genetically edited to overcome these obstacles, could improve remyelination. Here, we edit human(h) embryonic stem cell-derived OPCs to be unresponsive to a chemorepellent released from chronic MS lesions, and transplant them into rodent models of chronic lesions. Edited hOPCs display enhanced migration and remyelination compared to controls, regardless of the host age and length of time post-transplant. We show that genetic manipulation and transplantation of hOPCs overcomes the negative environment inhibiting remyelination, with translational implications for therapeutic strategies for people with progressive MS.

Gene editing of NCF1 loci is associated with homologous recombination and chromosomal rearrangements.

CRISPR-based genome editing of pseudogene-associated disorders, such as p47phox-deficient chronic granulomatous disease (p47 CGD), is challenged by chromosomal rearrangements due to presence of multiple targets. We report that interactions between highly homologous sequences that are localized on the same chromosome contribute substantially to post-editing chromosomal rearrangements. We successfully employed editing approaches at the NCF1 gene and its pseudogenes, NCF1B and NCF1C, in a human cell line model of p47 CGD and in patient-derived human hematopoietic stem and progenitor cells. Upon genetic engineering, a droplet digital PCR-based method identified cells with altered copy numbers, spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context, such as the presence of homologous regions, whose instability can constitute a risk factor for chromosomal rearrangements upon genome editing.

Biotechnological studies towards improvement of finger millet using multi-omics approaches.

A plethora of studies have uncovered numerous important genes with agricultural significance in staple crops. However, when it comes to orphan crops like minor millet, genomic research lags significantly behind that of major crops. This situation has promoted a focus on exploring research opportunities in minor millets, particularly in finger millet, using cutting-edge methods. Finger millet, a coarse cereal known for its exceptional nutritional content and ability to withstand environmental stresses represents a promising climate-smart and nutritional crop in the battle against escalating environmental challenges. The existing traditional improvement programs for finger millet are insufficient to address global hunger effectively. The lack of utilization of high-throughput platforms, genome editing, haplotype breeding, and advanced breeding approaches hinders the systematic multi-omics studies on finger millet, which are essential for pinpointing crucial genes related to agronomically important and various stress responses. The growing environmental uncertainties have widened the gap between the anticipated and real progress in crop improvement. To overcome these challenges a combination of cutting-edge multi-omics techniques such as high-throughput sequencing, speed breeding, mutational breeding, haplotype-based breeding, genomic selection, high-throughput phenotyping, pangenomics, genome editing, and more along with integration of deep learning and artificial intelligence technologies are essential to accelerate research efforts in finger millet. The scarcity of multi-omics approaches in finger millet leaves breeders with limited modern tools for crop enhancement. Therefore, leveraging datasets from previous studies could prove effective in implementing the necessary multi-omics interventions to enrich the genetic resource in finger millet.

Revolutionizing soybean genomics: How CRISPR and advanced sequencing are unlocking new potential.

Soybean Glycine max L., paleopolyploid genome, poses challenges to its genetic improvement. However, the development of reference genome assemblies and genome sequencing has completely changed the field of soybean genomics, allowing for more accurate and successful breeding techniques as well as research. During the single-cell revolution, one of the most advanced sequencing tools for examining the transcriptome landscape is single-cell RNA sequencing (scRNA-seq). Comprehensive resources for genetic improvement of soybeans may be found in the SoyBase and other genomics databases. CRISPR-Cas9 genome editing technology provides promising prospects for precise genetic modifications in soybean. This method has enhanced several soybean traits, including as yield, nutritional value, and resistance to both biotic and abiotic stresses. With base editing techniques that allow for precise DNA modifications, the use of CRISPR-Cas9 is further increased. With the availability of the reference genome for soybeans and the following assembly of wild and cultivated soybeans, significant chromosomal rearrangements and gene duplication events have been identified, offering new perspectives on the complex genomic structure of soybeans. Furthermore, major single nucleotide polymorphisms (SNPs) linked to stachyose and sucrose content have been found through genome-wide association studies (GWAS), providing important tools for enhancing soybean carbohydrate profiles. In order to open up new avenues for soybean genetic improvement, future research approaches include investigating transcriptional divergence processes, enhancing genetic resources, and incorporating CRISPR-Cas9 technologies.