CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.

Pancreatic cancer is marked by a poor prognosis and an exceptionally high mortality rate, with its aggressive nature contributing to its classification as a highly malignant disease. For effective therapeutic strategies, the development of sophisticated and regulated DNA manipulation methods is essential. Originally part of the prokaryotic immune system, CRISPR/Cas has emerged as a pivotal genome-editing tool with promising applications in pancreatic cancer research and therapy. This gene editing method is known for simplicity, rapid advancement, and superior precision compared to earlier techniques. Its adaptability allows precise gene editing for therapeutic purposes, including oncogene silencing and correction of pathogenic mutations. Additionally, CRISPR-driven gene editing has facilitated the development of pancreatic cancer models, which serve as valuable platforms for drug discovery and personalized treatment strategies, offering deeper insights into the genetic landscape of pancreatic tumors. This article provides an overview of the current applications of CRISPR technology in gene therapy and cancer research, particularly in the context of pancreatic cancer, and lays the foundation for future studies.

Gene editing provides precise, heritable genome mutagenesis without permanent transgenesis, and has been widely demonstrated and applied in planta. In the past decade, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) has revolutionized the application of gene editing in crops, with mechanistic advances expanding its potential, including prime editing and base editing. To date, CRISPR/Cas has been utilized in over a dozen orphan crops with diverse genetic backgrounds, leading to novel alleles and beneficial phenotypes for breeders, growers, and consumers. In conjunction with the adoption of science-based regulatory practices, there is potential for CRISPR/Cas-mediated gene editing in orphan crop improvement programs to solve a plethora of agricultural problems, especially impacting developing countries. Genome sequencing has progressed, becoming more affordable and applicable to orphan crops. Open-access resources allow for target gene identification and guide RNA (gRNA) design and evaluation, with modular cloning systems and enzyme screening methods providing experimental feasibility. While the genomic and mechanistic limitations are being overcome, crop transformation and regeneration continue to be the bottleneck for gene editing applications. International collaboration between all stakeholders involved in crop improvement is vital to provide equitable access and bridge the scientific gap between the world’s most economically important crops and the most under-researched crops. This review describes the mechanisms and workflow of CRISPR/Cas in planta and addresses the challenges, current applications, and future prospects in orphan crops.

RNA interference (RNAi) is a conserved molecular mechanism that plays a critical role in post-transcriptional gene silencing across diverse organisms. This review delves into the role of RNAi in plant functional genomics and its applications in crop improvement, highlighting its mechanistic insights and practical implications. The overview starts with the fundamental finding of the mechanism of RNAi, following its journey from petunias to its ubiquitous presence in a wide range of species. The discovery of several kinds of regulatory non-coding small RNAs, including as phasiRNAs, miRNAs, and siRNAs, has broadened our knowledge of RNAi-mediated gene regulation beyond what is previously thought. These RNA types have an impact on gene expression through complex post-transcriptional and epigenetic mechanisms. RNA interference (RNAi) has become a potent tool for comprehending gene activities in the context of crop improvement. It has shown to be useful in determining the functions of genes for metabolic pathways, stress tolerance, and more. Furthermore, RNAi-based strategies have potential for sustainable agriculture and integrated pest management, supporting international efforts to ensure food security. The many uses of RNA interference (RNAi) are covered in this article, including improving crop nutrition, changing plant architecture, and increasing shelf life. The challenges and future prospects of RNAi technology are also explored. The worldwide geography of RNA interference research is emphasized, with notable contributions from North America, Europe, and China. In conclusion, RNA interference (RNAi) continues to be a flexible and essential tool in contemporary plant research, providing new pathways for understanding gene activities and enhancing crop characteristics. Its combination with other biotechnological techniques, such as gene editing, might influence how agriculture and sustainable food production develop in the future.

Pre-B-Cell Leukemia Transcription Factor 1 Regulates Expression of Valosin-Containing Protein, a Gene Involved in Cancer Growth

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria-based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up-to-date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful.

Abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) technology is currently one of the most promising gene editing techniques. Gene-editing techniques allow various alterations to the DNA sequence by either addition, deletion, or inversion. The two primary components of this technique are the Cas9 endonuclease, which cuts the DNA strands at the specific target position of the genome, and the guide RNA molecule (gRNA), which guides the Cas9 endonuclease to that target portion. This technology is based on the adaptive immune system in prokaryotes, which prevents the entry of viruses by integrating short virus sequences in the cell’s CRISPR locus and allowing it to remember, recognize, and clear infections. The use of CRISPR technology in cancer biology is evolving quickly and holds great promise for the development of cancer models, blocking drug resistance, screening functional genes, gene editing, and CAR T cell therapy.

Abstract. The CRISPR-Cas gene editing system has revolutionized molecular biology by providing a versatile and precise method for genetic manipulation. We explore the basic mechanisms of CRISPR-Cas technology, focusing on the role of Cas proteins and guide RNAs in targeting and cleaving specific DNA sequences. Various types of CRISPR-Cas systems are also discussed, focusing on the widely used CRISPR-Cas9, and their applications in genome editing, including gene knockout, gene knock-in, and gene regulation. Additionally, we present advances in CRISPR-Cas technologies, such as base editing and plasmid editing, demonstrating their potential for therapeutic applications. This paper aims to provide a comprehensive overview of the status and applications of CRISPR-Cas technology and its future directions in gene editing.

Advances in gene editing now allow reverse genetics to be applied to a broad range of biological systems. Ultimately, any modification to coding sequences requires confirmation at the protein level, although immunoblotting is often hampered by antibody quality or availability especially in non-model species. Sequential Window Acquisition of All Theoretical Spectra (SWATH), a mass spectrometry (MS) technology with exceptional quantitative reproducibility and accuracy, offers an ideal alternative for protein-based confirmation. Here, using genome edits in mouse, zebrafish and Bicyclus anynana butterflies produced using either homologous recombination or targeted nucleases, we demonstrate absence of the targeted proteins using SWATH, thus confirming successful editing. We show that SWATH is a robust antibody-independent alternative for monitoring gene editing at the protein level and broadly applicable across diverse organisms and targeted genome manipulation techniques. Moreover, SWATH concomitantly defines the global proteome response in the edited organism, which may provide pertinent biological insights.

Hyaluronan (HA), a natural high molecular weight polysaccharide, has extensive applications in cosmetology and medical treatment. Hyaluronan-degrading enzymes (Hyals) act as molecular scissors that cleave HA by breaking the glucosidic linkage. Hyals are present in diverse organisms, including vertebrates, invertebrates and microorganisms, and play momentous roles in biological processes. In recent years, microbial Hyals (mHyals) have gained considerable attention for their exceptional performance in the production and processing of HA. Moreover, the applications of mHyals have been greatly extended to various biomedical fields. To explore the potential applications of mHyals, a thorough comprehension is imperative. In this context, this review systematically summarizes the sources, structures, mechanisms and enzymatic properties of mHyals and discusses their biological functions in host invasion, disease development, and regulation of intestinal flora. Furthermore, versatile applications inspired by their biological functions in medicine development, molecular biology, and industrial biotechnology are comprehensively reviewed. Finally, prospects are presented to emphasize the importance of exploration, expression and characterization of mHyals and the necessity of excavating their potential in biotechnological fields.Graphical abstract

Emerging Gene-editing nano-therapeutics for Cancer

Extensive industrialization and urbanization have led to pollution and the release of toxic material into soil and air. Most of the conventional methods for removal of metal pollutant are time-consuming, labor-, and capital- intensive which makes them uneconomical and hence can be effectively replaced by natural, eco-friendly methods such as phytoremediation. Systematic knowledge of plant–microbe interaction along with the use of gene editing and gene manipulation may increase the phytoremediation efficiency of plants. There exist several gene editing techniques and clustered regularly interspaced palindromic repeats (CRISPR) which are revolutionary tools that enable gene editing and can alter targeted traits in plants. CRISPR-mediated genome editing can be used in gene regulation by repression, and activation is of paramount importance in phytoremediation. CRISPR-mediated genome editing enhances phytostabilization, phytoextraction, phytomining, phytovolatilization, and bioenergy generation

ABSTRACTThe recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system expands the genome editing toolbox. This system exhibits several distinct features compared to the widely used CRISPR-Cas9 system, but has reduced gene editing efficiency. To optimize the CRISPR-Cpf1 (Cas12a) system, we report the inclusion of self-cleaving ribozymes that facilitate processing of the crRNA transcript to produce the precise guide molecule. Insertion of the 3ʹ-terminal HDV ribozyme boosted the gene editing activity of the CRISPR-Cpf1 system ranging from 1.1 to 5.2 fold. We also demonstrate that this design can enhance CRISPR-based gene activation. We thus generated an improved CRISPR-Cpf1 system for more efficient gene editing and gene regulation.

Histone methyltransferase absent, small, or homeotic discs1-like (ASH1L) is composed of su(var)3-9, enhancer of zeste, trithorax (SET) domain, pleckstrin homology domain (PHD) domain, middle (MID) domain, and bromo adjacent homology (BAH) domain. The SET domain of ASH1L is known to mediate mediate H3K36 dimethylation (H3K36me2) modification. However, the specific functions of the PHD-BAH domain remain largely unexplored. This study aimed to explore the biological function of the PHD-BAH domain in ASH1L. We employed a range of techniques, including a prokaryotic fusion protein expression purification system, pull-down assay, Isothermal Titration Calorimetry (ITC), polymerase chain reaction (PCR), and sitedirected mutagenesis, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) gene editing, cell culture experiment, western blot, cell proliferation assay, and cell apoptosis test. The PHD-BAH domain in ASH1L preferentially binds to the H3K4me2 peptide over H3K4 monomethylation (H3K4me1) and H3K4 trimethylation (H3K4me3) peptide. Notably, the W2603A mutation within the PHD-BAH domain could disrupt the interaction with H3K4me2 in vitro. Compared with wild-type Cholangiocarcinoma (CHOL) cells, deletion of the PHD-BAH domain in ASH1L led to increased CHOL cell apoptosis and reduced cell proliferation (P < 0.001). Additionally, the W2603A mutation affected the regulation of the proteasome 20S subunit beta (PSMB) family gene set. W2603A mutation was crucial for the interaction between the PHD-BAH domain and the H3K4me2 peptide. ASH1L regulated CHOL cell survival and proliferation through its PHD-BAH domain by modulating the expression of the PSMB family gene set.

DIPA-CRISPR is a simple and accessible method for insect gene editing

Abalone, a marine mollusk with significant economic and ecological value, plays a crucial role in sustainable aquaculture. The development and application of CRISPR-Cas9 gene-editing technology have opened up a new path for improving breeding efficiency. CRISPR/Cas9-mediated gene editing has been achieved in abalones via microinjection. In this study, a gene encoding myostatin MSTN in H. discus hannai; was selected as target for conducting the CRISPR-Cas9 gene editing experiment in combination with an electroporation delivery system. Our results showed that all three sgRNAs effectively targeted and cleaved the target segment, with sgRNA1 and sgRNA2 exhibiting high in vitro activity. After electroporation, the effects of transfection on embryonic development of fertilized eggs were observed and statistically analyzed. 12.7 ± 5.4% of the fertilized eggs were damaged and deformed after electroporation. Twenty-four hours after electroporation, surviving larvae were collected for DNA extraction and sequencing. Two potential mutations within the target region of MSTN were identified by sequencing. These results provide a reference for the improvement and development of CRISPR-mediated gene editing methods in marine mollusks such as abalones.