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Found: 26 180
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Genome engineering with Cas9 and AAV repair templates, successes and pitfalls.
Genome editing, in particular the CRISPR/Cas9 system, is widely used to generate new animal models. However, the generation of mutations, such as conditional knock-out or knock-in, can remain complex and inefficient, in particular because of the difficulty to deliver the donor DNA (single or double stranded) into the nucleus of fertilized oocytes. The use of recombinant adeno-associated viruses (rAAV) as donor DNA is a rapidly developing approach that promises to improve the efficiency of creation of animal models. In this mini-review, we explore the progress and challenges of using CRISPR/Cas9 in combination with rAAV for precise genome editing. We will summarise the current knowledge of rAAV transduction, data on its use in rodent embryos in combination with CRISPR/Cas9 to easily generate sequence replacements or insertions, the limitations of rAAV and the unexpected events observed to date, and the protocol optimisations already in place to facilitate its use in the generation of animal models.
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CRISPRCasStack: a stacking strategy-based ensemble learning framework for accurate identification of Cas proteins.
CRISPR-Cas system is an adaptive immune system widely found in most bacteria and archaea to defend against exogenous gene invasion. One of the most critical steps in the study of exploring and classifying novel CRISPR-Cas systems and their functional diversity is the identification of Cas proteins in CRISPR-Cas systems. The discovery of novel Cas proteins has also laid the foundation for technologies such as CRISPR-Cas-based gene editing and gene therapy. Currently, accurate and efficient screening of Cas proteins from metagenomic sequences and proteomic sequences remains a challenge. For Cas proteins with low sequence conservation, existing tools for Cas protein identification based on homology cannot guarantee identification accuracy and efficiency. In this paper, we have developed a novel stacking-based ensemble learning framework for Cas protein identification, called CRISPRCasStack. In particular, we applied the SHAP (SHapley Additive exPlanations) method to analyze the features used in CRISPRCasStack. Sufficient experimental validation and independent testing have demonstrated that CRISPRCasStack can address the accuracy deficiencies and inefficiencies of the existing state-of-the-art tools. We also provide a toolkit to accurately identify and analyze potential Cas proteins, Cas operons, CRISPR arrays and CRISPR-Cas locus in prokaryotic sequences. The CRISPRCasStack toolkit is available at https://github.com/yrjia1015/CRISPRCasStack.
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High-efficiency breeding of early-maturing rice cultivars via CRISPR/Cas9-mediated genome editing.
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Are we ready to genetically modify a human embryo? Or is it too late to ask?
After the announcement that a scientist genetically altered human embryos and implanted them for gestation last November, the increased discussion has revolved around the ethical lapses that occurred in the process. The scientist, He Jiankui, forged ethical approval documents, chose a disease target that is preventable and treatable, and did not replicate a naturally occurring mutation, creating a new mutation instead. Aside from these and other ethical concerns about his procedure and protocol, several fundamental questions remain: should scientists be altering the human germline at this time and, if so, under what circumstances? If not, under what circumstances might such research be permissible, if at all? And who should make these decisions? We believe that it is too early for this research to be conducted in embryos intended for gestation. Additional safety and efficacy research on animal and non-implanted embryos is needed. But more importantly, a broad societal conversation should inform decisions about the future of germline editing, including determining the scope of relevant ethical considerations, such as the conditions under which the research is ready for human application (if at all), and the oversight such research requires. Abbreviations: First-in-Human (FIH); National Academies of Science, Engineering, and Medicine (NASEM).
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Precision breeding in agriculture and food systems in the United Kingdom.
In recent years there have been major advances in precision breeding technologies, such as gene editing, that offer promising solutions to revolutionise global crop production and tackle the pressing issues in food systems. The UK has leading expertise in genomics, and research is already taking place to develop crops with improved resilience to climate change, resistance to disease and less reliance on chemical inputs. In March 2023, the Genetic Technology (Precision Breeding) Act received Royal Assent and passed into UK law. It provides a framework from which to build more proportionate regulations for plants and animals made using genetic technologies which contain genetic changes that could also arise through traditional breeding-known as 'Precision Bred Organisms'. New legislation and the utilization of UK world-leading research could help to enhance the efficiency of breeding systems and enable the development of plants and animals that are healthier, better for the environment and more resilient to climate change.
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Prevalent integration of genomic repetitive and regulatory elements and donor sequences at CRISPR-Cas9-induced breaks.
CRISPR-Cas9 genome editing has been extensively applied in both academia and clinical settings, but its genotoxic risks, including large insertions (LgIns), remain poorly studied due to methodological limitations. This study presents the first detailed report of unintended LgIns consistently induced by different Cas9 editing regimes using various types of donors across multiple gene loci. Among these insertions, retrotransposable elements (REs) and host genomic coding and regulatory sequences are prevalent. RE frequencies and 3D genome organization analysis suggest LgIns originate from randomly acquired genomic fragments by DNA repair mechanisms. Additionally, significant unintended full-length and concatemeric double-stranded DNA (dsDNA) donor integrations occur when donor DNA is present. We further demonstrate that phosphorylated dsDNA donors consistently reduce large insertions and deletions by almost two-fold without compromising homology-directed repair (HDR) efficiency. Taken together, our study addresses a ubiquitous and overlooked risk of unintended LgIns in Cas9 editing, contributing valuable insights for the safe use of Cas9 editing tools.
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CRISPR/Cas12a-mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus.
Purple non-sulfur photosynthetic bacteria (PNSB) such as Rhodobacter capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for genome editing and transcriptional regulation in R. capsulatus. Template-free disruption method mediated by CRISPR/Cas12a reached ˜ 90% editing efficiency when targeting ccoO or nifH gene. When both genes were simultaneously edited, the multiplex editing efficiency reached > 63%. In addition, CRISPR interference (CRISPRi) using deactivated Cas12a was also evaluated using reporter genes egfp and lacZ, and the transcriptional repression efficiency reached ˜ 80%. In summary, our work represents the first report to develop CRISPR/Cas12a-mediated genome editing and transcriptional regulation in R. capsulatus, which would greatly accelerate PNSB-related researches.
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Possible Reversion of CRISPR-Cas9-Edited Sequences in Octoploid Strawberry.
Gene editing is more challenging in octoploids due to the presence of multiple copies of each gene. However, the ability to edit genes in these plants would allow editing in commercial varieties. Here, we delivered sequences targeting FaMYB9 into octoploid strawberry "Honeoye" and identified several gene-edited lines. Among them, the heterozygous gene-edited line FaMYB9CR-15 had curved and wrinkled leaves at 3 months, whereas leaves of 3-month-old wild-type (WT) strawberry seedlings were elliptical with a smooth surface. At that stage, FaMYB9CR-15 leaves also had large patches of wax. We identified 11,402 differentially expressed genes, divided into four clusters, between WT and FaMYB9CR-15 seedlings at 3 months. Notably, cluster 4 genes-related to nonhomologous end joining, microhomology-mediated end joining repairs, homologous recombination, nucleotide excision repair, and mismatch repair-were more highly expressed in the gene-edited line than in the WT. Surprisingly, by 6 months of age, FaMYB9CR-15 leaves had become smooth with small patches of wax, and expression levels of cluster 4 genes were significantly lower than at 3 months. Over the same period, the percentage of FaMYB9 loci harboring the mutant allele decreased from 70.2% to 43.7%. These findings lead us to conclude that there could be reversion of mutated sequences in octoploid strawberry, emphasizing the challenges of gene editing high-ploidy materials.
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HLA-G gene editing in tumor cell lines as a novel alternative in cancer immunotherapy.
Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial.W In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G - cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.
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pegIT - a web-based design tool for prime editing.
Prime editing (PE) is a novel CRISPR-derived genome editing technique facilitating precision editing without double-stranded DNA breaks. PE, mediated by a Cas9-reverse transcriptase fusion protein, is based on dual-functioning prime editing guide RNAs (pegRNAs), serving both as guide molecules and as templates carrying the desired edits. Due to such diverse functions, manual pegRNA design is a subject to error and not suited for large-scale setups. Here, we present pegIT, a user-friendly web tool for rapid pegRNA design for numerous user-defined edits, including large-scale setups. pegIT is freely available at https://pegit.giehmlab.dk.
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