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Combination of ssDNA recombineering and CRISPR-Cas9 for Pseudomonas putida KT2440 genome editing.
Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon-based plasmid cloned with the tightly regulated redβ and cas9 genes; the second is a broad host range, pBBR1 replicon-based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.
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Epigenetic editing works like a CHARM.
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CRISPR-cas9-Mediated Gene Editing to Reverse Oncogenic Mutations in Oral Squamous Cell Carcinoma.
INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a genetically driven malignancy characterized by a high burden of oncogenic mutations that contribute to aggressive tumor behavior, therapeutic resistance, and poor survival outcomes. Conventional treatment modalities largely target downstream molecular pathways without correcting the underlying genetic aberrations, underscoring the need for precision-based therapeutic strategies. This study aimed to assess the feasibility and functional impact of CRISPR-Cas9-mediated gene editing in reversing oncogenic mutations associated with OSCC using an in vitro experimental model. MATERIALS AND METHODS: A controlled in vitro experimental study was conducted using the established human OSCC cell lines harboring mutations in key oncogenic genes. Cells were divided into control, mock-transfected, and CRISPR-Cas9-edited groups. Target-specific single-guide RNAs were designed to correct oncogenic mutations using advanced CRISPR-based editing platforms. Gene-editing efficiency was validated by molecular assays, while functional outcomes were assessed using cell proliferation analysis. Statistical evaluation was done using the one-way ANOVA with significance set at P < 0.05. RESULTS: CRISPR-Cas9-edited OSCC cells demonstrated a significant reduction in cell proliferation compared to control and mock-transfected groups ( P < 0.001). No significant variation was found between control and mock-transfected cells, confirming minimal procedural influence. CONCLUSION: CRISPR-Cas9-mediated correction of oncogenic mutations effectively suppresses malignant cellular proliferation in OSCC, highlighting its promising role as a precision therapeutic strategy in oral cancer management.
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Bacterial Retrons Enable Precise Gene Editing in Human Cells.
Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve the efficiency of precise genome editing via homology-directed repair (HDR). In human cells, HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.4%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.
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For China, a CRISPR first goes too far.
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Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing.
Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called 'BALL', enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.
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Sharpening the Scissors: Mechanistic Details of CRISPR/Cas9 Improve Functional Understanding and Inspire Future Research.
Interest in CRISPR/Cas9 remains high level as new applications of the revolutionary gene-editing tool continue to emerge. While key structural and biochemical findings have illuminated major steps in the enzymatic mechanism of Cas9, several important details remain unidentified or poorly characterized that may contribute to known functional limitations. Here we describe the foundation of research that has led to a fundamental understanding of Cas9 and address mechanistic uncertainties that restrict continued development of this gene-editing platform, including specificity for the protospacer adjacent motif, propensity for off-target binding and cleavage, as well as interactions with cellular components during gene editing. Discussion of these topics and considerations should inspire future research to hone this remarkable technology and advance CRISPR/Cas9 to new heights.
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A review of CRISPR associated genome engineering: application, advances and future prospects of genome targeting tool for crop improvement.
The Cas9 nuclease initiates double-stranded breaks at the target position in DNA, which are repaired by the intracellular restoration pathways to eliminate or insert pieces of DNA. CRISPR-Cas9 is proficient and cost-effective since cutting is guided by a piece of RNA instead of protein. Emphasis on this technology, in contrast with two recognized genome editing platforms (i.e., ZFNs and TALENs), is provided. This review evaluates the benefits of chemically synthesized gRNAs as well as the integration of chemical amendments to improve gene editing efficiencies. CRISPR is an indispensable means in biological investigations and is now as well transforming varied fields of biotechnology and agriculture. Recent advancement in targetable epigenomic-editing tools allows researchers to dispense direct functional and transcriptional significance to locus-explicit chromatin adjustments encompassing gene regulation and editing. An account of diverse sgRNA design tools is provided, principally on their target competence prediction model, off-target recognition algorithm, and generation of instructive annotations. The modern systems that have been utilized to deliver CRISPR-Cas9 in vivo and in vitro for crop improvement viz. nutritional enhancement, production of drought-tolerant and disease-resistant plants, are also highlighted. The conclusion is focused on upcoming directions, biosafety concerns, and expansive prospects of CRISPR technologies.
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Human Germline Gene Editing from Maslahah Perspective: The Case of the World's First Gene Edited Babies.
This paper describes maslahah, a fundamental concept in Islam and its application in deliberating permissibility of human germline gene editing from an Islamic perspective. This paper refers to He Jiankui's research that led to the birth of the world's first gene edited babies, who were edited to be protected from HIV. The objective, procedure, and output of the research were assessed against the conditions of maslahah. It can be concluded that the experiment did not meet the conditions; it is inconsistent with the objectives of shariah (maqasid al-shariah) and some fundamental Islamic teachings that it did not preserve greater benefit, hence it could be considered impermissible.
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Enrichment of Allelic Editing Outcomes by Prime Editing in Induced Pluripotent Stem Cells.
Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase and PE guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay at the HEK3 benchmark locus and at the APOE Alzheimer's risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes.
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