Golden Gate Cloning of Synthetic CRISPR RNA Spacer Sequences.

Prokaryotes use CRISPR-Cas systems to interfere with viruses and other mobile genetic elements. CRISPR arrays comprise repeated DNA elements and spacer sequences that can be engineered for custom target sites. These arrays are transcribed into precursor CRISPR RNAs (pre-crRNAs) that undergo maturation steps to form individual CRISPR RNAs (crRNAs). Each crRNA contains a single spacer that identifies the target cleavage site for a large variety of Cas protein effectors. Precise manipulation of spacer sequences within CRISPR arrays is crucial for advancing the functionality of CRISPR-based technologies. Here, we describe a protocol for the design and creation of a minimal, plasmid-based CRISPR array to enable the expression of specific, synthetic crRNAs. Plasmids contain entry spacer sequences with two type IIS restriction sites and Golden Gate cloning enables the efficient exchange of these spacer sequences. Factors that influence the compatibility of the CRISPR arrays with native or recombinant Cas proteins are discussed.

Enhancement of fermentation traits in industrial Baker's yeast for low or high sugar environments.

Saccharomyces cerevisiae SPC-SNU 70-1 is a commercial diploid baking yeast strain valued for its excellent bread-making qualities, including superior leavening capabilities and the production of flavor-enhancing volatile organic acids. Despite its benefits, this strain faces challenges in fermenting both lean (low-sugar) and sweet (high-sugar) doughs. To address these issues, we employed the CRISPR/Cas9 genome editing system to modify genes without leaving any genetic scars. For lean doughs, we enhanced the yeast's ability to utilize maltose over glucose by deleting a gene involved in glucose repression. For sweet doughs, we increased glycerol production by overexpressing glycerol biosynthetic genes and optimizing redox balance, thereby improving the tolerence to osmotic stress during fermentation. Additionally, the glycerol-overproducing strain demonstrated enhanced freeze tolerance, and bread made from this strain exhibited improved storage properties. This study demonstrates the feasibility and benefits of using engineered yeast strains, created solely by editing their own genes without introducing foreign genes, to enhance bread making.

Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.

Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells. As an example, we describe how to knock out the glutamine synthetase (GS) gene, which increases the selection efficiency of the GS-mediated gene amplification system.

CRISPR applications in microbial World: Assessing the opportunities and challenges.

Genome editing has emerged during the past few decades in the scientific research area to manipulate genetic composition, obtain desired traits, and deal with biological challenges by exploring genetic traits and their sequences at a level of precision. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a genome editing tool has offered a much better understanding of cellular and molecular mechanisms. This technology emerges as one of the most promising candidates for genome editing, offering several advantages over other techniques such as high accuracy and specificity. In the microbial world, CRISPR/Cas technology enables researchers to manipulate the genetic makeup of micro-organisms, allowing them to achieve almost impossible tasks. This technology initially discovered as a bacterial defense mechanism, is now being used for gene cutting and editing to explore more of its dimensions. CRISPR/Cas 9 systems are highly efficient and flexible, leading to its widespread uses in microbial research areas. Although this technology is widely used in the scientific community, many challenges, including off-target activity, low efficiency of Homology Directed Repair (HDR), and ethical considerations, still need to be overcome before it can be widely used. As CRISPR/Cas technology has revolutionized the field of microbiology, this review article aimed to present a comprehensive overview highlighting a brief history, basic mechanisms, and its application in the microbial world along with accessing the opportunities and challenges.

Lipopeptide-mediated Cas9 RNP delivery: A promising broad therapeutic strategy for safely removing deep-intronic variants in ABCA4.

Deep-intronic (DI) variants represent approximately 10%-12% of disease-causing genetic defects in ABCA4-associated Stargardt disease (STGD1). Although many of these DI variants are amenable to antisense oligonucleotide-based splicing-modulation therapy, no treatment is currently available. These molecules are mostly variant specific, limiting their applicability to a broader patient population. In this study, we investigated the therapeutic potential of the CRISPR-Cas9 system combined with the amphipathic lipopeptide C18:1-LAH5 for intracellular delivery to correct splicing defects caused by different DI variants within the same intron. The combination of these components facilitated efficient editing of two target introns (introns 30 and 36) of ABCA4 in which several recurrent DI variants are found. The partial removal of these introns did not affect ABCA4 splicing or its expression levels when assessed in two different human cellular models: fibroblasts and induced pluripotent stem cell-derived photoreceptor precursor cells (PPCs). Furthermore, the DNA editing in STGD1 patient-derived PPCs led to a ∼50% reduction of the pseudoexon-containing transcripts resulting from the c.4539+2001G>A variant in intron 30. Overall, we provide proof-of-concept evidence of the use of C18:1-LAH5 as a delivery system for therapeutic genome editing for ABCA4-associated DI variants, offering new opportunities for clinical translation.

Integration of CRISPR/Cas9 with multi-omics technologies to engineer secondary metabolite productions in medicinal plant: Challenges and Prospects.

Plants acts as living chemical factories that may create a large variety of secondary metabolites, most of which are used in pharmaceutical products. The production of these secondary metabolites is often much lower. Moreover, the primary constraint after discovering potential metabolites is the capacity to manufacture sufficiently for use in industrial and therapeutic contexts. The development of omics technology has brought revolutionary discoveries in various scientific fields, including transcriptomics, metabolomics, and genome sequencing. The metabolic pathways leading to the utilization of new secondary metabolites in the pharmaceutical industry can be identified with the use of these technologies. Genome editing (GEd) is a versatile technology primarily used for site-directed DNA insertions, deletions, replacements, base editing, and activation/repression at the targeted locus. Utilizing GEd techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9), metabolic pathways engineered to synthesize bioactive metabolites optimally. This article will briefly discuss omics and CRISPR/Cas9-based methods to improve secondary metabolite production in medicinal plants.

CRISPR: New promising biotechnological tool in wastewater treatment.

The increasing demand for water resources with increase in population has sparked interest in reusing produced water, especially in water-scarce regions. The clustered regularly interspaced short palindromic repeats (CRISPR) technology is an emerging genome editing tool that has the potential to trigger significant impact with broad application scope in wastewater treatment. We provide a comprehensive overview of the scope of CRISPR-Cas based tool for treating wastewater that may bring new scope in wastewater management in future in controlling harmful contaminants and pathogens. As an advanced versatile genome engineering tool, focusing on particular genes and enzymes that are accountable for pathogen identification, regulation of antibiotic/antimicrobial resistance, and enhancing processes for wastewater bioremediation constitute the primary focal points of research associated with this technology. The technology is highly recommended for targeted mutations to incorporate desirable microalgal characteristics and the development of strains capable of withstanding various wastewater stresses. However, concerns about gene leakage from strains with modified genome and off target mutations should be considered during field application. A comprehensive interdisciplinary approach involving various fields and an intense research focus concerning delivery systems, target genes, detection, environmental conditions, and monitoring at both lab and ground level should be considered to ensure its successful application in sustainable and safe wastewater treatment.

Preventive Human Genome Editing and Enhancement: Candidate Criteria for Governance.

While somatic cell editing to treat disease is widely accepted, the use of human genome editing for "enhancement" remains contested. Scientists and policy-makers routinely cite the prospect of enhancement as a salient ethical challenge for human genome editing research. If preventive genome editing projects are perceived as pursuing human enhancement, they could face heightened barriers to scientific, public, and regulatory approval. This article outlines what we call "preventive strengthening research" (or "PSR") to explore, through this example, how working to strengthen individuals' resistance to disease beyond what biomedicine considers to be the human functional range may be interpreted as pursuing human enhancement. Those involved in developing guidance for PSR will need to navigate the interface between preventive goals and enhancement implications. This article identifies and critiques three of these ideas in the interest of anticipating the wider emergence of PSR and the need for a normative approach for its pursuit. All three "candidate criteria" merit attention, but each also faces challenges that will need to be addressed as further research policy is developed.

Proximity-dependent biotinylation reveals an interaction between ubiquitin-specific peptidase 46 and centrosome-related proteins.

Protein ubiquitination extensively modulates protein functions and controls various biological processes, such as protein degradation, signal transduction, transcription, and DNA repair. Ubiquitination is a reversible post-translational modification, and deubiquitinating enzymes cleave ubiquitin from proteins. Ubiquitin-specific peptidase 46 (USP46), a deubiquitinase, is highly expressed in the brain and regulates neural functions. Deleting lysine 92 (ΔK92) in USP46 reduces murine depression-like behavior in the tail suspension test. However, the molecular basis for USP46's role in regulating neural function has not yet been fully understood. Here we employed a proximity-dependent biotinylation approach to characterize the USP46 protein interaction partners. Using homology-independent targeted integration (HITI), a genome editing technique, we established knockin cell lines that stably express USP46 wildtype- or ΔK92-biotin ligase fusion protein. We identified 286 candidate interaction partners, including well-known binding partners of USP46. Although there were no obvious differences in the interactome of USP46 between wildtype and ΔK92, a gene ontology analysis revealed that centrosome-related proteins were significantly enriched in the proximal proteins of USP46. Several centrosome-related proteins were bound to USP46 in Neuro2a cells, but their protein expression levels were not affected in the brains of USP46-deficient mice. These results uncover a potential relationship between USP46 and centrosome regulation independently of protein stabilization.

CRISPR/Cas technology: fueling the future of Biofuel production with sugarcane.

The objective of present review is to provide a scientific overview of sugarcane as a potential feedstock for biofuel and use of genome editing approach for improvement of industrial and agronomical traits in sugarcane. Sugarcane, a perennial tropical grass with a high biomass index, is a promising feedstock for bioethanol production, and its bagasse, rich in lignocellulosic material, serves as an ideal feedstock for producing second-generation bioethanol. To improve the conversion of sugarcane biomass into biofuels, developing varieties with improved biomass degradability and high biomass and sucrose content is essential. The complex genome architecture and earlier lack of sequence data hindered biotechnological advancements in sugarcane, but recent genome sequence updates offer new opportunities for sugarcane improvement. The first genetically modified sugarcane was developed in 1992 by Bower and Birch using microprojectile bombardment of embryogenic callus. Since then, transgenic techniques have rapidly evolved, leading to the advancement of genome editing technologies. Application of genome editing tools particularly CRISPR/Cas system has been successfully used in sugarcane for editing. Recently, multiple alleles of the magnesium chelatase and acetolactate synthase genes in sugarcane have been successfully edited through multiplexing. Additionally, CRISPR-edited sugarcane varieties with modified cell wall components and increased sucrose content for enhanced bioethanol production have been developed. At the end, the future of CRISPR-edited crops will depend on how well regulatory frameworks adapt to the rapidly evolving technology.

Balanced Training Sets Improve Deep Learning-Based Prediction of CRISPR sgRNA Activity.

CRISPR-Cas systems have transformed the field of synthetic biology by providing a versatile method for genome editing. The efficiency of CRISPR systems is largely dependent on the sequence of the constituent sgRNA, necessitating the development of computational methods for designing active sgRNAs. While deep learning-based models have shown promise in predicting sgRNA activity, the accuracy of prediction is primarily governed by the data set used in model training. Here, we trained a convolutional neural network (CNN) model and a large language model (LLM) on balanced and imbalanced data sets generated from CRISPR-Cas12a screening data for the yeast Yarrowia lipolytica and evaluated their ability to predict high- and low-activity sgRNAs. We further tested whether prediction performance can be improved by training on imbalanced data sets augmented with synthetic sgRNAs. Lastly, we demonstrated that adding synthetic sgRNAs to inherently imbalanced CRISPR-Cas9 data sets from Y. lipolytica and Komagataella phaffii leads to improved performance in predicting sgRNA activity, thus underscoring the importance of employing balanced training sets for accurate sgRNA activity prediction.

Efficient gene disruption in polyploid genome by Cas9-Trex2 fusion protein.

The fusion of the exonuclease Trex2 with the Cas9 protein significantly enhanced the efficiency of genome editing in hexaploid common wheat, particularly for the simultaneous editing of multiple favorable alleles within a single generation, thereby facilitating genome editing-assisted breeding in polyploid crops.

CRISPR/Cas9 mediated genome editing for crop improvement against Abiotic stresses: current trends and prospects.

Abiotic stresses associated with climate change, such as heat, cold, salinity, and drought, represent a serious threat to crop health. To mitigate the risks posed by these environmental challenges, both transgenic technology and conventional breeding methods have been extensively utilized. However, these methods have faced numerous limitations. The development of synthetic nucleases as precise genetic tools allows for the targeted alteration of stress-responsive genes in crop improvement. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas) genome-editing technique has transformed gene editing with its broad applicability, accessibility, adaptability, flexibility, and simplicity. Its application shows promise for the development of crop types that are more able to survive abiotic stress conditions. The present study presents recent scenario and application of CRISPR/Cas genome-editing technology in enhancing crop tolerance to a variety of abiotic stresses.

CRISPR/Cas system-mediated base editing in crops: recent developments and future prospects.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) genome-editing system has altered plant research by allowing for targeted genome alteration, and they are emerging as powerful tools for evaluating plant gene function and improving crop yield. Even though CRISPR/Cas9 cleavage and subsequent repair are effective ways to precisely replace genes and change base pairs in plants, the dominance of the non-homologous end-joining pathway (NHEJ) and homology-directed repair's (HDR) poor effectiveness in plant cells have restricted their use. Base editing is gaining popularity as a potential alternative to HDR or NHEJ-mediated replacement, allowing for precise changes in the plant genome via programmed conversion of a single base to another without the need for a donor repair template or double-stranded breaks. In this review, we primarily present the mechanisms of base-editing system, including their distinct types such as DNA base editors (cytidine base editor and adenine base editor) and RNA base editors discovered so far. Next, we outline the current potential applications of the base-editing system for crop improvements. Finally, we discuss the limitations and potential future directions of the base-editing system in terms of improving crop quality. We hope that this review will enable the researcher to gain knowledge about base-editing tools and their potential applications in crop improvement.

Strategies for improving the genome-editing efficiency of class 2 CRISPR/Cas system.

Since its advent, gene-editing technology has been widely used in microorganisms, animals, plants, and other species. This technology shows remarkable application prospects, giving rise to a new biotechnological industry. In particular, third-generation gene editing technology, represented by the CRISPR/Cas9 system, has become the mainstream gene editing technology owing to its advantages of high efficiency, simple operation, and low cost. These systems can be widely used because they have been modified and optimized, leading to notable improvements in the efficiency of gene editing. This review introduces the characteristics of popular CRISPR/Cas systems and optimization methods aimed at improving the editing efficiency of class 2 CRISPR/Cas systems, providing a reference for the development of superior gene editing systems. Additionally, the review discusses the development and optimization of base editors, primer editors, gene activation and repression tools, as well as the advancement and refinement of compact systems such as IscB, TnpB, Fanzor, and Cas12f.

An efficient CRISPR-Cas12a-mediated MicroRNA knockout strategy in plants.

In recent years, the CRISPR-Cas9 nuclease has been used to knock out MicroRNA (miRNA) genes in plants, greatly promoting the study of miRNA function. However, due to its propensity for generating small insertions and deletions, Cas9 is not well-suited for achieving a complete knockout of miRNA genes. By contrast, CRISPR-Cas12a nuclease generates larger deletions, which could significantly disrupt the secondary structure of pre-miRNA and prevent the production of mature miRNAs. Through the case study of OsMIR390 in rice, we confirmed that Cas12a is a more efficient tool than Cas9 in generating knockout mutants of a miRNA gene. To further demonstrate CRISPR-Cas12a-mediated knockout of miRNA genes in rice, we targeted nine OsMIRNA genes that have different spaciotemporal expression and have not been previously investigated via genetic knockout approaches. With CRISPR-Cas12a, up to 100% genome editing efficiency was observed at these miRNA loci. The resulting larger deletions suggest Cas12a robustly generated null alleles of miRNA genes. Transcriptome profiling of the miRNA mutants, as well as phenotypic analysis of the rice grains revealed the function of these miRNAs in controlling gene expression and regulating grain quality and seed development. This study established CRISPR-Cas12a as an efficient tool for genetic knockout of miRNA genes in plants.

Geminiviruses and Food Security: A Molecular Genetics Perspective for Sustainable Agriculture in Africa.

The African continent is vulnerable to food insecurity. Increased food costs, job losses, and climate change force Africans to chronic hunger. Biotechnology can be used to mitigate this by using techniques such as CRISPR/Cas9 systems, TALENs, and ZFNs. Biotechnology can utilize geminiviruses to deliver the necessary reagents for precise genome alteration. Additionally, plants infected with geminiviruses can withstand harsher weather conditions such as drought. Therefore, this article discusses geminivirus replication and its use as beneficial plant DNA viruses. It focuses explicitly on genome editing to increase plant resistance by manipulating plants' salicylic acid and jasmonic acid pathways.

CRISPR-based functional genomics for schistosomes and related flatworms.

CRISPR genome editing is actively used for schistosomes and other flukes. The ability to genetically manipulate these flatworms enables deeper investigation of their (patho)biological nature. CRISPR gene knockout (KO) demonstrated that a liver fluke growth mediator contributes to disease progression. Genome safe harbor sites have been predicted in Schistosoma mansoni and targeted for transgene insertion. CRISPR-based diagnosis has been demonstrated for infection with schistosomes and Opisthorchis viverrini. This review charts the progress, and the state of play, and posits salient questions for the field to address. Derivation of heritably transgenic loss-of-function or gain-of-function lines is the next milestone.

CRISPR/Cas9 technology for advancements in cancer immunotherapy: from uncovering regulatory mechanisms to therapeutic applications.

In recent years, immunotherapy has developed rapidly as a new field of tumour therapy. However, the efficacy of tumour immunotherapy is not satisfactory due to the immune evasion mechanism of tumour cells, induction of immunosuppressive tumour microenvironment (TME), and reduction of antigen delivery, etc. CRISPR/Cas9 gene editing technology can accurately modify immune and tumour cells in tumours, and improve the efficacy of immunotherapy by targeting immune checkpoint molecules and immune regulatory genes, which has led to the great development and application. In current clinical trials, there are still many obstacles to the application of CRISPR/Cas9 in tumour immunotherapy, such as ensuring the accuracy and safety of gene editing, overcoming overreactive immune responses, and solving the challenges of in vivo drug delivery. Here we provide a systematic review on the application of CRISPR/Cas9 in tumour therapy to address the above existing problems. We focus on CRISPR/Cas9 screening and identification of immunomodulatory genes, targeting of immune checkpoint molecules, manipulation of immunomodulators, enhancement of tumour-specific antigen presentation and modulation of immune cell function. Second, we also highlight preclinical studies of CRISPR/Cas9 in animal models and various delivery systems, and evaluate the efficacy and safety of CRISPR/Cas9 technology in tumour immunotherapy. Finally, potential synergistic approaches for combining CRISPR/Cas9 knockdown with other immunotherapies are presented. This study underscores the transformative potential of CRISPR/Cas9 to reshape the landscape of tumour immunotherapy and provide insights into novel therapeutic strategies for cancer patients.

Membrane progesterone receptor γ (paqr5b) is essential for the formation of neurons in the zebrafish olfactory rosette.

Paqr5b is a gene encoding membrane progesterone receptor γ (mPRγ), which is one of five mPR subtypes. Paqr5b belongs to the progestin and adipoQ receptor (PAQR) family, which consists of 11 genes. To elucidate the physiological functions of the mPR subtypes, we established gene knockout (KO) zebrafish strains by genetically editing seven paqr genes and analyzed their phenotypes. The null-mutant strain of paqr5b (paqr5b-/-) that we established in this study showed low fecundity, reduced chorion elevation and a high percentage of abnormal embryos. Embryos showed curvature of the spine and an abnormal head morphology. Individuals with abnormal head morphology continued to develop a phenotype of markedly abnormal palatine bone. The length of the brain of paqr5b-/- zebrafish was short, and the position of the cerebellum moved to the front and overlapped with that of the midbrain. Micro-CT scans revealed that the olfactory rosettes (ORs) were so shrunken that they were difficult to identify and connected with the olfactory bulbs (OBs) by thread-like structures. Immunohistochemical staining of OR with an anti-Paqr5b antibody revealed that Paqr5b was extensively expressed in neurons in the OR in wild-type zebrafish, whereas signals were not detected in paqr5b-/- zebrafish. In histological sections, the neurons disappeared, and the lamellar layer of the OR became thinner. These results indicate that Paqr5b is required for the formation of neurons in the OR. This is the first report demonstrating a distinct role for the mPR gene.