Clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized creating targeted genetic variation in crops. Although CRISPR enzymes have been reported to have high sequence-specificity, careful design of the editing reagents can also reduce unintended edits at highly homologous sites. This work details the first large-scale study of the heritability of on-target edits and the rate of edits at off-target sites in soybean (Glycine max), assaying ~700 T1 plants each resulting from transformation with LbCas12a constructs containing CRISPR RNAs (crRNAs) predicted to be either \"unique\" with no off-target sites or \"promiscuous\" with >10 potential off-targets in the soybean genome. Around 80% of the on-target edits observed in T0 plants were inherited in the T1 generation, and ~49% of the total observed on-target edits in T1 were not observed at T0, indicating continued activity of LbCas12a throughout the life cycle of the plant. In planta editing at off-target sites was observed for the Promiscuous but not the Unique crRNA. Examination of the edited off-target sites revealed that LbCas12a was highly tolerant to mismatches between the crRNA and target site in bases 21-23 relative to the start of the protospacer, but even a single mismatch in the first 20 nt drastically reduced the editing rate. In addition, edits at off-target sites have lower inheritance rates than on-target edits, suggesting that they occur later in the plant\'s lifecycle. Plants with a desired on-target edit and no off-target edits could be identified in the T1 generation for 100% of the T0 plants edited with the Unique crRNA compared with the 65% of T0 plants edited with the Promiscuous crRNA. This confirms that proper crRNA selection can reduce or eliminate off-target editing. Even when potential off-target sites are predicted, plants containing only the intended edits can still be identified and propagated.

UNASSIGNED: Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated canker-resistant Citrus sinensis cv. Hamlin lines in the T0 generation. This was achieved through the transformation of embryogenic protoplasts using the ribonucleoprotein (RNP) containing Cas12a and one crRNA to edit the canker susceptibility gene, CsLOB1, which led to small indels.
UNASSIGNED: Here, we transformed embryogenic protoplasts of Hamlin with RNP containing Cas12a and three crRNAs.
UNASSIGNED: Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversions were observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions maybe required for inversions. Biallelic mutations were observed for each of the three target sites in four of the 10 edited lines when three crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a and three crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These cslob1 mutant lines were canker- resistant and no canker symptoms were observed after inoculation with Xcc and Xcc growth was significantly reduced in the cslob1 mutant lines compared to the wild type plants.
UNASSIGNED: Taken together, RNP (Cas12a and three crRNAs) transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments.

BACKGROUND: CRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community.
RESULTS: We identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately.
CONCLUSIONS: Based on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.

The global burden of cancer and the limitations of conventional therapies highlight the potential of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) in reshaping cancer treatment paradigms. In this review, we have investigated the mechanism of CRISPR, an adaptive immune system in bacteria that enables highly precise gene editing at the molecular level. This versatile tool demonstrates its efficacy in human cancer therapy through gene knockout, metabolic disruption, base editing, screening, and immunotherapy enhancement without affecting normal bodily domains. Despite its superiority over other nucleases like zinc-finger nucleases and transcription activator-like effector nucleases, hurdles such as off-target effects, inefficient delivery of the system to target cells, the emergence of escapers, and the ethical debate surrounding genome editing are discussed. In this article, we have reviewed the promising approaches of CRISPR-Cas9 in cancer treatment while exploring the underlying mechanism, advantages, and associated challenges.

Base editing represents a cutting-edge genome editing technique that utilizes the CRISPR system to guide base deaminases with high precision to specific genomic sites, facilitating the targeted alteration of individual nucleotides. Unlike traditional gene editing approaches, base editing does not require DNA double-strand breaks or donor templates. It functions independently of the cellular DNA repair machinery, offering significant advantages in terms of both efficiency and accuracy. In this review, we summarize the core design principles of various DNA base editors, their distinctive editing characteristics, and tactics to refine their efficacy. We also summarize their applications in crop genetic improvement and explore their potential contributions to forest genetic engineering.

IgM is the major circulating Ig isotype in teleost fish, showing in Antarctic fish unique features such as an extraordinary long hinge region, which plays a crucial role in antibody structure and function. In this work, we describe the replacement of the hinge region of a murine monoclonal antibody (mAb) with the peculiar hinge from Antarctic fish IgM. We use the CRISPR/Cas9 system as a powerful tool for generating the engineered mAb. Then, we assessed its functionality by using an innovative plasmonic substrate based on bimetallic nanoislands (AgAuNIs). The affinity constant of the modified mAb was 2.5-fold higher than that obtained from wild-type mAb against the specific antigen. Here, we show the suitability of the CRISPR/Cas9 method for modifying a precise region in immunoglobulin gene loci. The overall results could open a frontier in further structural modifications of mAbs for biomedical and diagnostic purposes.

The Pax6 gene is essential for eye and brain development across various animal species. Here, we investigate the function of Pax6 in the development of the anterior central nervous system (CNS) of the invertebrate chordate amphioxus using CRISPR/Cas9-induced genome editing. Specifically, we examined Pax6 mutants featuring a 6 bp deletion encompassing two invariant amino acids in the conserved paired domain, hypothesized to impair Pax6 DNA-binding capacity and gene regulatory functions. Although this mutation did not result in gross morphological changes in amphioxus larvae, it demonstrated a reduced ability to activate Pax6-responsive reporter gene, suggesting a hypomorphic effect. Expression analysis in mutant larvae revealed changes in gene expression within the anterior CNS, supporting the conserved role of Pax6 gene in brain regionalization across chordates. Additionally, our findings lend support to the hypothesis of a zona limitans intrathalamica (ZLI)-like region in amphioxus, suggesting evolutionary continuity in brain patterning mechanisms. ZLI region, found in both hemichordates and vertebrates, functions as a key signaling center and serves as a restrictive boundary between major thalamic regions.

Gene manipulation of hematopoietic stem cells (HSCs) using the CRISPR/Cas system as a potent genome editing tool holds immense promise for addressing hematologic disorders. An essential hurdle in advancing this treatment lies in effectively delivering CRISPR/Cas to HSCs. While various delivery formats exist, Ribonucleoprotein complex (RNP) emerges as a particularly efficient option. RNP complexes offer enhanced gene editing capabilities, devoid of viral vectors, with rapid activity and minimized off-target effects. Nevertheless, novel delivery methods such as microfluidic-based techniques, filtroporation, nanoparticles, and cell-penetrating peptides are continually evolving. This study aims to provide a comprehensive review of these methods and the recent research on delivery approaches of RNP complexes to HSCs.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system continues to evolve, thereby enabling more precise detection and repair of mutagenesis. The development of CRISPR/Cas-based diagnosis holds promise for high-throughput, cost-effective, and portable nucleic acid screening and genetic disease diagnosis. In addition, advancements in transportation strategies such as adeno-associated virus (AAV), lentiviral vectors, nanoparticles, and virus-like vectors (VLPs) offer synergistic insights for gene therapeutics in vivo. Wilson\'s disease (WD), a copper metabolism disorder, is primarily caused by mutations in the ATPase copper transporting beta (ATP7B) gene. The condition is associated with the accumulation of copper in the body, leading to irreversible damage to various organs, including the liver, nervous system, kidneys, and eyes. However, the heterogeneous nature and individualized presentation of physical and neurological symptoms in WD patients pose significant challenges to accurate diagnosis. Furthermore, patients must consume copper-chelating medication throughout their lifetime. Herein, we provide a detailed description of WD and review the application of novel CRISPR-based strategies for its diagnosis and treatment, along with the challenges that need to be overcome.

Among plant-derived secondary metabolites are benzylisoquinoline alkaloids (BIAs) that play a vital role in medicine. The most conspicuous BIAs frequently found in opium poppy are morphine, codeine, thebaine, papaverine, sanguinarine, and noscapine. BIAs have provided abundant clinically useful drugs used in the treatment of various diseases and ailments With an increasing demand for these herbal remedies, genetic improvement of poppy plants appears to be essential to live up to the expectations of the pharmaceutical industry. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9), the field of metabolic engineering has undergone a paradigm shift in its approach due to its appealing attributes, such as the transgene-free editing capability, precision, selectivity, robustness, and versatility. The potentiality of the CRISPR system for manipulating metabolic pathways in opium poppy was demonstrated, but further investigations regarding the use of CRISPR in BIA pathway engineering should be undertaken to develop opium poppy into a bioreactor synthesizing BIAs at the industrial-scale levels. In this regard, the recruitment of RNA-guided genome editing for knocking out miRNAs, flower responsible genes, genes involved in competitive pathways, and base editing are described. The approaches presented here have never been suggested or applied in opium poppy so far.