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Co-CRISPR: A valuable toolkit for mutation enrichment in the gene editing of Spodoptera frugiperda.
The CRISPR/Cas9 system has been successfully applied in dozens of diverse species; although the screening of successful CRISPR/Cas9 editing events remains particularly laborious, especially for those that occur at relatively low frequency. Recently, a co-CRISPR strategy was proved to enrich the desired CRISPR events. Here, the co-CRISPR strategy was developed in the Fall armyworm, Spodoptera frugiperda, with kynurenine 3-monooxygenase gene (kmo) as a marker. The kmo mosaics induced by single-guide RNAs (sgRNAs)/Cas9 displayed the darker green color phenotype in larvae, compared with wild type (brown), and mosaic-eye adults were significantly acquired from the mosaic larvae group. In the kmo knockout strain, no significant difference was observed in larval development and adult reproduction. Acetylcholinesterase 2 (ace2) and Wnt1 were selected as target genes to construct the co-CRISPR strategy using kmo marker. By co-injection of kmo and ace2 sgRNAs, the mutant efficiency of ace2 was significantly increased in the kmo mosaic (larvae or adults) groups. Similarly, more malformed pupae with Wnt1 mutations were observed in the darker green larvae group. Taken together, these results demonstrated that kmo was a suitable visible marker gene for the application and extension of co-CRISPR strategy in Fall armyworm. Using darker green color in larvae or mosaic-eye in adults from kmo knockout as a marker, the mutant efficiency of a target gene could be enriched in a Fall armyworm group consisting of marked individuals. The co-CRISPR strategy is helpful for gene function studies by the knockout technique with no or lethal phenotypes.
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Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice.
CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (>1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.
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Genetic Engineering of Human Keratinocytes Using CRISPR/Cas9 Ribonucleoprotein Complexes or Modified Cas9-Encoding mRNAs.
CRISPR/Cas9 is a straightforward genome-editing technique that is implemented across disciplines and research areas. However, in keratinocytes, CRISPR/Cas9 can be particularly difficult due to variable genome-editing efficiency, reduced cell viability, and difficulties during (sub)cloning of gene-edited keratinocyte populations. Here, we provide a step-by-step detailed protocol for the genetic manipulation of human (primary) keratinocytes, including widely accepted procedures for the analysis of CRISPR/Cas9 efficiency, (sub)cloning procedures to select heterozygous or homozygous keratinocytes, and off-target genome-editing analysis.
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Delivery of genome editors with engineered virus-like particles.
Genome editing technologies have revolutionized biomedical sciences and biotechnology. However, their delivery in vivo remains one of the major obstacles for clinical translation. Here, we introduce various emerging genome editing systems and review different delivery systems have been developed to realize the promise of in vivo gene editing therapies. In particular, we focus on virus-like particles (VLPs), an emerging delivery platform and provide in depth analysis on recent advancements to improve VLPs delivery potential and highlight opportunities for future improvements. To this end, we also provide detail workflows for engineered VLP (eVLP) selection, production, and purification, along with methods for characterization and validation.
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Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells.
The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.
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Cas9 Ribonucleoproteins (RNPs) for Gene Deletion.
Genetic manipulations in pathogenic microorganisms, like Aspergillus fumigatus, allow us to study the function of genes in pathogenicity or other important traits. Classic genetic engineering tools in filamentous fungi rely on DNA fragment sub-cloning or fusion PCR to build a gene deletion cassette containing extended flanking regions of homology to promote efficient recombination. However, the introduction of CRISPR technology into fungal genetics has greatly simplified and accelerated the genome editing process. Here, we describe a simple and universal, one-step CRISPR-Cas9-mediated genetic tool employing repair templates containing microhomology regions. This gene targeting system displays high homologous recombination efficiency rates and can be easily implemented across different genetic backgrounds.
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CRISPR-Cas9 claim sets and the potential to stifle innovation.
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Genome Editing of Mouse by Cytoplasmic Injection.
CRISPR/Cas enables rapid production of genome-edited animals. The Cas9/gRNA component can be introduced to fertilized eggs in several ways. Here, we provide an instructional guide for the generation of knockout mice by cytoplasmic injection using in vitro transcribed Cas9 and gRNA.
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PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells.
Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%-30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.
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Innovative Chemical Strategies for Advanced CRISPR Modulation.
Over the past decade, RNA-guided gene editing technologies, particularly those derived from CRISPR systems, have revolutionized life sciences and opened unprecedented opportunities for therapeutic innovation. Despite their transformative potential, achieving precise control over the activity and specificity of these molecular tools remains a formidable challenge, requiring advanced and innovative regulatory strategies. We and others have developed new approaches that integrate chemical ingenuity with bioorthogonal techniques to achieve remarkable precision in CRISPR regulation. One key innovation lies in the chemical modulation of guide RNA (gRNA), significantly expanding the CRISPR toolkit. Strategies such as CRISPR-ON and CRISPR-OFF switches rely on selective chemical masking and demasking of gRNA. These approaches use either bulky chemical groups to preemptively mask RNA or minor, less obstructive groups to fine-tune its function, followed by bioorthogonal reactions to restore or suppress activity. These methodologies have proven to be pivotal for controlled gene editing and expression, addressing the challenges of precision, reversibility, and dynamic regulation.Parallel to these advances, the development of mesoporous metal-organic frameworks (MOFs) has emerged as a promising solution for RNA deprotection and activation. By serving as catalytic tools, MOFs enhance the versatility and efficiency of CRISPR systems, pushing their applications beyond the conventional boundaries. In addition, the synthesis of novel small molecules for regulating CRISPR-Cas9 activity marks a critical milestone in the evolution of gene therapy protocols. Innovative RNA structural control strategies have also emerged, particularly through the engineering of G-quadruplex (G4) motifs and G-G mismatches. These methods exploit the structural propensities of engineered gRNAs, employing small-molecule ligands to induce specific conformational changes that modulate the CRISPR activity. Whether stabilizing G4 formation or promoting G-G mismatches, these strategies demonstrate the precision and sophistication required for the molecular-level control of gene editing.Further enhancing these innovations, techniques like host-guest chemistry and conditional diacylation cross-linking have been developed to directly alter gRNA structure and function. These approaches provide nuanced, reversible, and safe control over CRISPR systems, advancing both the precision and reliability of gene editing technologies. In conclusion, this body of work highlights the convergence of chemistry, materials science, and molecular biology to create integrative solutions for gene editing. By combination of bioorthogonal chemistry, RNA engineering, and advanced materials, these advancements offer unprecedented accuracy and control for both fundamental research and therapeutic applications. These innovations not only advance genetic research but also contribute to developing safer and more effective gene editing strategies, moving us closer to realizing the full potential of these technologies.
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