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Targeting Specificity of the CRISPR/Cas9 System.
CRISPR/Cas9 system has accelerated research across many fields since its demonstration for genome editing. CRISPR also offers vast therapeutic potential, but an important hurdle of this technology is the off-target mutations it can induce. In this viewpoint, we will discuss recent strategies for improving CRISPR specificity, emphasizing how a complete mechanistic understanding of CRISPR/Cas9 can benefit such efforts. We also propose that agreeing upon a consensus protocol with the highest specificity could benefit researchers working on CRISPR-based therapies. In addition to improving CRISPR/Cas9 specificity, accurate detection of off-target events is also crucial, and we will discuss various unbiased off-target detection methods in terms of their advantages and disadvantages. We suggest that using a combination of cell-based and cell-free methods can prove more useful. In addition, we point out that improving predictive algorithms for off-target sites would require pooling of the available off-target analysis data and standardization of the protocols used for obtaining the data. Moreover, we highlight the risk of insertional mutagenesis for gene correction applications requiring the use of donor DNA. We conclude by discussing future prospects for the field, as well as steps that can be taken to overcome the aforementioned challenges.
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Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing.
The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.
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Genetic modification of the ant Lasius niger using CRISPR-Cas9 technology.
CRISPR-Cas9 has become one of the most prominent gene editing tools available and it has been utilized in various organisms from bacteria to fungi, plants, and animals. In this study, we developed a CRISPR-Cas9 protocol for the black garden ant Lasius niger, a common and easily available study species for lab and field experiments. To create indel mutations using CRISPR-Cas9 in L. niger, we targeted three different locations in a well-studied eye pigmentation gene cinnabar, generating several mutations that disrupt the ommochrome biosynthesis pathway and result in the lack of the pigment and therefore, abnormal eye coloration in adult workers. We also developed a protocol to collect L. niger eggs, inject them with CRISPR-Cas9 construct, and rear the eggs into mature adult workers with the assistance of nursing workers. We demonstrated for the first time in L. niger that CRISPR-Cas9 is an excellent tool to create targeted mutations for this species. Our protocol can be referred to when developing similar studies for other species of ants and eusocial insects.
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Optimizing Rice Genomics: Employing the Hypercompact Cas12j2 System for Targeted Transcriptional Regulation and Epigenome Modification.
In the burgeoning field of genome engineering, the CRISPR-Cas systems have emerged as pivotal tools for precise genetic modifications in various organisms, including humans, animals, and plants. One significant obstacle in this arena is the substantial size of Cas proteins, such as SpCas9, which is approximately 190 kDa, complicating their delivery, particularly via viral vectors. To overcome this challenge, our research introduces the hypercompact Cas12j2 system, a groundbreaking development with a size of merely ~80 kDa, originally identified in Biggiephage. We demonstrate its application in plant genome editing, with a particular focus on rice. In this context, we have successfully adapted Cas12j2 for gene activation, achieving significant increases in gene expression, specifically up to a tenfold activation for OsER1 and a fourfold activation for OsNRT1.1A in stable transgenic rice plants. Moreover, we have ventured beyond mere gene editing to develop a Cas12j2-based approach for targeted epigenome editing, particularly in the context of DNA methylation. This was demonstrated through the targeted methylation of the OsGBSS1 promoter, as verified by Next-Generation Sequencing of bisulfite sequencing PCR products. This chapter presents a detailed protocol about utilizing the hypercompact Cas12j2 system in conjunction with specific effectors, such as transcriptional activation or repression domains, or methylation domains, to achieve targeted gene transcriptional regulation and epigenome modification in rice.
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Keeping CRISPR in check.
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A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity.
The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the pathway with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes.
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Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles.
CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5' handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2'-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2'-hydroxyl sensitivity. Modified 5' pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5' pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5' pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.
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Muscle-specific gene editing therapy via mammalian fusogen-directed virus-like particles.
Muscle genetic defects can lead to impaired movement, respiratory failure, and other severe symptoms. The development of curative therapies is challenging due to the need for the delivery of gene-editing tools into skeletal muscle cells throughout the body. Here, we use muscular fusogens (Myomaker and Myomerger) to engineer muscle-specific virus-like particles (MuVLPs) for the systemic delivery of gene-editing tools. We demonstrate that MuVLPs can be loaded with diverse payloads, including EGFP, Cre and Cas9/sgRNA ribonucleoproteins (Cas9 RNPs), and can be delivered into skeletal muscle cells via targeted membrane fusion. Systemic administration of MuVLPs carrying Cas9 RNPs enables skeletal muscle-specific gene editing, which excised the exon containing a premature terminator codon mutation in a mouse model for Duchenne muscular dystrophy (DMD). This treatment restores dystrophin expression in various skeletal muscle tissues, including the diaphragm, quadriceps, tibialis anterior, gastrocnemius, and triceps. As a result, the treated mice exhibit a significantly increased capacity for exercise and endurance. This study established a platform for precise gene editing in skeletal muscle tissues.
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Lipid nanoparticle-mediated CRISPR/Cas9 delivery enables efficient trabecular meshwork gene editing in mice.
Lipid nanoparticles (LNPs) enable efficient mRNA delivery, yet their potential for ocular gene editing remains largely unexplored. Here, we systematically evaluated three LNP formulations containing distinct ionizable lipids, DLin-MC3-DMA, ALC0315, and SM102, for gene delivery to ocular tissues. Among them, SM102-based LNP encapsulating GFP mRNA (SM102-GFP) exhibited the highest transfection efficiency across three cultured ocular cells in vitro. Following intravitreal injection in mice, SM102-GFP achieved selective and robust expression in the trabecular meshwork (TM) without detectable retinal transfection. GFP expression in TM peaked at one week post-injection, declined by three weeks, and could be effectively re-induced by a second dosing of the same vector. Compared with adeno-associated viral (AAV) and adenoviral (Ad) vectors, SM102-GFP showed superior TM specificity and reduced retinal inflammation. Co-delivery of SpCas9 mRNA and sgRNA via SM102-based LNPs enabled efficient CRISPR-mediated knockout of Matrix Gla Protein (Mgp), a key inhibitor of TM calcification. Mgp knockout induced sustained intraocular pressure elevation and anterior chamber deepening with open angles, recapitulating features of primary open-angle glaucoma. Chronic ocular hypertension further led to Müller gliosis and ganglion cell complex thinning, indicative of progressive retinal stress. These findings establish SM102-based LNPs as a safe and efficient platform for TM-targeted gene editing and glaucoma modeling.
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CRISPR/Cas9 Genome Editing in the Diamondback Moth: Current Progress, Challenges, and Prospects.
The development of site-specific genome-editing tools like CRISPR (clustered regularly interspaced short palindromic repeat) and its associated protein, Cas9, is revolutionizing genetic engineering with its highly efficient mechanism, offering the potential for effective pest management. Recently, CRISPR/Cas9 gene-editing has been extensively utilized in the management of the diamondback moth, Plutella xylostella (L.), a highly destructive pest of vegetable crops, for different purposes, such as gene function analysis and genetic control. However, the progress related to this gene-editing tool in P. xylostella has not yet been summarized. This review highlights the progress and applications of CRISPR/Cas9 in uncovering the genes critical for development, reproduction, and insecticide resistance in P. xylostella. Moreover, the progress related to the CRISPR/Cas9 gene drive for population suppression and modifications has also been discussed. In addition to the significant progress made, challenges such as low germline editing efficiency and limited homology-directed repair remain obstacles to its widespread application. To address these limitations, we have discussed the different strategies that are anticipated to improve the efficiency of CRISPR/Cas9, paving the way to it becoming a pivotal tool in sustainable pest management. Therefore, the present review will help researchers in the future enhance the efficiency of the CRISPR/Cas9 system and use it to manage the diamondback moth.
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