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Gold Nanomaterials-Implemented CRISPR-Cas Systems for Biosensing.
Due to their superiority in the simple design and precise targeting, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have attracted significant interest for biosensing. On the one hand, CRISPR-Cas systems have the capacity to precisely recognize and cleave specific DNA and RNA sequences. On the other hand, CRISPR-Cas systems such as orthologs of Cas9, Cas12, and Cas13 exhibit cis-cleavage or trans-cleavage activities after recognizing the target sequence. Owing to the cleavage activities, CRISPR-Cas systems can be designed for biosensing by degrading tagged nucleic acids to produce detectable signals. To meet the requirements of point-of-care detection and versatile signal readouts, gold nanomaterials with excellent properties such as high extinction coefficients, easy surface functionalization, and biocompatibility are implemented in CRISPR-Cas-based biosensors. In combination with gold nanomaterials such as gold nanoparticles, gold nanorods, and gold nanostars, great efforts are devoted to fabricating CRISPR-Cas-based biosensors for the detection of diverse targets. This review focuses on the current advances in gold nanomaterials-implemented CRISPR-Cas-based biosensors, particularly the working mechanism and the performance of these biosensors. CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13a are discussed and highlighted. Meanwhile, prospects and challenges are also discussed in the design of biosensing strategies based on gold nanomaterials and CRISPR-Cas systems.
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Targeted Genetic Changes in Candida albicans Using Transient CRISPR-Cas9 Expression.
Candida albicans is an opportunistic fungal pathogen responsible for significant disease and mortality. Absent complete mating and other convenient methods, dissection of its virulence factors relies on robust tools to delete, complement, and otherwise modify genes of interest in this diploid organism. Here we describe the design principles and use of CRISPR associated nuclease 9 (Cas9) and single-guide RNAs transiently expressed from PCR cassettes to modify genes of interest, generating homozygous mutants in a single transformation step. © 2021 Wiley Periodicals LLC. Basic Protocol 1: PCR amplification of CRISPR components Basic Protocol 2: Transformation of Candida albicans Basic Protocol 3: Selecting and genotyping transformants Alternate Protocol 1: Deletion with recyclable markers by CRISPR induced marker excision (CRIME) Alternate Protocol 2: Knock-in and combining multiple cassettes with overlapping homology.
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How CRISPR/Cas9 Gene Editing Is Revolutionizing T Cell Research.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows for precise gene targeting in mammalian cells, including T cells, allowing scientists to disrupt or edit specific genes of interest. This has enabled immunologists to investigate T cell functions as well as opened the path for novel therapeutics involving gene editing of T cells ex vivo before transferring these back to patients to increase T cell efficacy. This review outlines how CRISPR/Cas9 has transformed T cell research allowing immunologists to rapidly probe the roles of genes in T cells thus paving the way for novel therapeutics. Furthermore, this review describes how these tools reduce the requirement for genetic mouse models, while increasing the translational potential of T cell research.
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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9-mediated mutagenesis of the multiple edematous wings gene induces muscle weakness and flightlessness in Bactrocera dorsalis (Diptera: Tephritidae).
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system is a versatile, efficient and heritable gene editing tool that can be useful for genome engineering. Bactrocera dorsalis (Hendel) is a major pest of agriculture that causes great economic losses. We used the B. dorsalis multiple edematous wings (Bdmew) gene as the target gene to explore the effectiveness of CRISPR/Cas9 for B. dorsalis genome manipulation. We studied the physiological functions of the Bdmew gene, particularly those related to muscle development. Site-specific genome editing was feasible using direct microinjection of specific guide RNA and the Cas9-plasmid into B. dorsalis embryos. Mutation frequencies ranged from 12.1 to 30.2% in the injected generation. Mosaic G0, with the mew mutation, was heritable to the next generation. The G1 displayed a series of defective phenotypes including muscle weakness, flightlessness, failure to eclose, wing folds and unbalanced movement. These results demonstrated that CRISPR/Cas9 can act as a highly specific, efficient, heritable tool for genome manipulation in B. dorsalis and this has significance for gene function research and genetic control of pests. The Bdmew gene possesses key functions in muscle development of B. dorsalis. Bdmew mutations cause a series of serious defects by interfering with muscle development and may provide a means for controlling B. dorsalis via a gene-based method such as gene drive.
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CRISPR-Cas-mediated targeted genome editing in human cells.
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems have evolved as an adaptive surveillance and defense mechanism in bacteria and archaea that uses short RNAs to direct degradation of foreign genetic elements. Here, we present our protocol for utilizing the S. pyogenes type II bacterial CRISPR system to achieve sequence-specific genome alterations in human cells. In principle, any genomic sequence of the form N₁₉NGG can be targeted with the generation of custom guide RNA (gRNA) which functions to direct the Cas9 protein to genomic targets and induce DNA cleavage. Here, we describe our methods for designing and generating gRNA expression constructs either singly or in a multiplexed manner, as well as optimized protocols for the delivery of Cas9-gRNA components into human cells. Genomic alterations at the target site are then introduced either through nonhomologous end joining (NHEJ) or through homologous recombination (HR) in the presence of an appropriate donor sequence. This RNA-guided editing tool offers greater ease of customization and synthesis in comparison to existing sequence-specific endonucleases and promises to become a highly versatile and multiplexable human genome engineering platform.
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The iCRISPR platform for rapid genome editing in human pluripotent stem cells.
Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. Here, we first describe the establishment of the iCRISPR platform through TALEN-mediated targeting of inducible Cas9 expression cassettes into the AAVS1 locus. Next, we provide a series of technical procedures for using iCRISPR to achieve one-step knockout of one or multiple gene(s), "scarless" introduction of precise nucleotide alterations, as well as inducible knockout during hPSC differentiation. We present an optimized workflow, as well as guidelines for the selection of CRISPR targeting sequences and the design of single-stranded DNA (ssDNA) homology-directed DNA repair templates for the introduction of specific nucleotide alterations. We have successfully used these protocols in four different hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic modifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease.
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A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants.
We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).
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Nucleic acid-guided genome editing of plant carbohydrate pathways for functional crops supporting glycogen replenishment in athletes.
Nucleic acid-guided genome editing is transforming plant breeding by enabling precise modification of metabolic pathways to meet evolving food and nutritional needs. In this study, we applied multiplex CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) editing to improve Ipomoea batatas (sweet potato), a climate-resilient staple, for enhanced starch yield and customized digestibility suited for athletic performance nutrition. We targeted four key starch-related genes: AGPase (ADP-glucose pyrophosphorylase), GBSSI (granule-bound starch synthase I), SBEII (starch branching enzyme II), and GPT1 (glucose-6-phosphate translocator). Editing efficiency reached 85 to 92 % across multiple genome copies (homeologs), with no detectable off-target effects. The edited lines showed up to a 20 % increase in total starch and a fivefold reduction in amylose, which is the slowly digestible fraction of starch. This resulted in a high-amylopectin profile associated with rapid glucose release. Transcriptome analysis confirmed the metabolic shifts, including increased expression of GPT1 and sucrose synthase and reduced expression of invertase, which supports improved carbon allocation toward starch biosynthesis. In vitro digestibility and glycemic index (GI) assessments confirmed the functional impact of these traits for glycogen replenishment. Yield performance and drought resilience were retained, confirming agronomic viability. This study highlights the potential of CRISPR-based editing to develop climate-smart, nutritionally functional crops for both health-focused applications and global food security.
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The CRISPR System and Cancer Immunotherapy Biomarkers.
Recent advances in cancer immunotherapy have shed new light on the possibility to cure most, if not all, cancer patients with further development of various treatment options. The emergency of a new genome editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR) technology, revolutionized the biomedical research field. We envision application of the CRISPR technology in cancer research, diagnosis, and therapy will markedly speed up the development of new treatment options for cancer patients. The CRISPR system and its applications in biomedical research will be discussed with an emphasis on cancer immunotherapy and biomarker development.
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Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives.
The use of third-generation lentiviral vectors is now commonplace in most areas of basic biology. These systems provide a fast, efficient means for modulating gene expression, but experimental design needs to be carefully considered to minimize potential artefacts arising from off-target effects and other confounding factors. This review offers a starting point for those new to lentiviral-based vector systems, addressing the main issues involved with the use of lentiviral systems in vitro and outlines considerations which should be taken into account during experimental design. Factors such as selecting an appropriate system and controls, and practical titration of viral transduction are important considerations for experimental design. We also briefly describe some of the more recent advances in genome editing technology. TALENs and CRISPRs offer an alternative to lentivirus, providing endogenous gene editing with reduced off-target effects often at the expense of efficiency.
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