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Found: 26 168
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Pseudogene-Mediated Gene Conversion After CRISPR-Cas9 Editing Demonstrated by Partial CD33 Conversion with SIGLEC22P.
Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocyte cell line. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines, which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.
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Innovative approaches to combat antibiotic resistance: integrating CRISPR/Cas9 and nanoparticles against biofilm-driven infections.
The increasing prevalence of antibiotic-resistant bacterial infections is a major global health concern, with biofilms playing a key role in bacterial persistence and resistance. Biofilms provide a protective matrix that limits antibiotic penetration, enhances horizontal gene transfer, and enables bacterial survival in hostile environments. Conventional antimicrobial therapies are often ineffective against biofilm-associated infections, necessitating the development of novel therapeutic strategies. The CRISPR/Cas9 gene-editing system has emerged as a revolutionary tool for precision genome modification, offering targeted disruption of antibiotic resistance genes, quorum sensing pathways, and biofilm-regulating factors. However, the clinical application of CRISPR-based antibacterials faces significant challenges, particularly in efficient delivery and stability within bacterial populations. Nanoparticles (NPs) present an innovative solution, serving as effective carriers for CRISPR/Cas9 components while exhibiting intrinsic antibacterial properties. Nanoparticles can enhance CRISPR delivery by improving cellular uptake, increasing target specificity, and ensuring controlled release within biofilm environments. Recent advances have demonstrated that liposomal CRISPR-Cas9 formulations can reduce Pseudomonas aeruginosa biofilm biomass by over 90% in vitro, while gold nanoparticle carriers enhance editing efficiency up to 3.5-fold compared to non-carrier systems. These hybrid platforms also enable co-delivery with antibiotics, producing synergistic antibacterial effects and superior biofilm disruption. Additionally, they can facilitate co-delivery of antibiotics or antimicrobial peptides, further enhancing therapeutic efficacy. This review explores the synergistic integration of CRISPR/Cas9 and nanoparticles in combating biofilm-associated antibiotic resistance. We discuss the mechanisms of action, recent advancements, and current challenges in translating this approach into clinical practice. While CRISPR-nanoparticle hybrid systems hold immense potential for next-generation precision antimicrobial therapies, further research is required to optimize delivery platforms, minimize off-target effects, and assess long-term safety. Understanding and overcoming these challenges will be critical for developing effective biofilm-targeted antibacterial strategies.
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Expanding the scope of genome editing with SpG and SpRY variants in rice.
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Generation of genetically tailored porcine liver cancer cells by CRISPR/Cas9 editing.
Pigs provide a valuable large animal model for several diseases due to their similarity with humans in anatomy, physiology, genetics and drug metabolism. We recently generated a porcine model for TP53R167H and KRASG12D driven hepatocellular carcinoma (HCC) by autologous liver implantation. Here we describe a streamlined approach for developing genetically tailored porcine HCC cells by CRISPR/Cas9 gene editing and isolation of homogenous genetically validated cell clones. The combination of CRISPR/Cas9 editing of HCC cells described herein with the orthotopic HCC model enables development of various porcine HCC models, each with a specific mutational profile. This allows modeling the effect of different driver mutation combinations on tumor progression and in vivo testing of novel targeted therapeutic approaches in a clinically relevant large animal model.
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Clipping Chlamy Genes: Improved Methods for Targeted Gene Editing in Chlamydomonas.
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Generation of genetically modified rat models via the CRISPR/Cas9 technology.
The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.
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Engineering Cas9 for human genome editing.
Since the initial reports describing CRISPR-Cas9, labs across the globe have leveraged this valuable gene editing tool to alter the genomes of living cells. With the goal of generating more precise and efficient genome changes, scientists and engineers have mutated, evolved, and covalently altered Cas9 in order to predictably edit the genetic code. Here, we highlight recent advancements and contributions to the growing field of Cas9 engineering. We present key aspects of Cas9 engineering efforts focused on sgRNA manipulation, PAM-recognition, specificity, deaminase fusions, reverse-transcriptase fusions, and structural rearrangements of this important gene-modifying tool.
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A Comparison of Techniques to Evaluate the Effectiveness of Genome Editing.
Genome editing using engineered nucleases (meganucleases, zinc finger nucleases, transcription activator-like effector nucleases) has created many recent breakthroughs. Prescreening for efficiency and specificity is a critical step prior to using any newly designed genome editing tool for experimental purposes. The current standard screening methods of evaluation are based on DNA sequencing or use mismatch-sensitive endonucleases. They can be time-consuming and costly or lack reproducibility. Here, we review and critically compare standard techniques with those more recently developed in terms of reliability, time, cost, and ease of use.
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Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration.
Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly reduced from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.
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A Versatile Nonviral Delivery System for Multiplex Gene-Editing in the Liver.
Recent advances in CRISPR present attractive genome-editing toolsets for therapeutic strategies at the genetic level. Here, a liposome-coated mesoporous silica nanoparticle (lipoMSN) is reported as an effective CRISPR delivery system for multiplex gene-editing in the liver. The MSN provides efficient loading of Cas9 plasmid as well as Cas9 protein/guide RNA ribonucleoprotein complex (RNP), while liposome-coating offers improved serum stability and enhanced cell uptake. Hypothesizing that loss-of-function mutation in the lipid-metabolism-related genes pcsk9, apoc3, and angptl3 would improve cardiovascular health by lowering blood cholesterol and triglycerides, the lipoMSN is used to deliver a combination of RNPs targeting these genes. When targeting a single gene, the lipoMSN achieved a 54% gene-editing efficiency, besting the state-of-art Lipofectamine CRISPRMax. For multiplexing, lipoMSN maintained significant gene-editing at each gene target despite reduced dosage of target-specific RNP. By delivering combinations of targeting RNPs in the same nanoparticle, synergistic effects on lipid metabolism are observed in vitro and vivo. These effects, such as a 50% decrease in serum cholesterol after 4 weeks of post-treatment with lipoMSN carrying both pcsk9 and angptl3-targeted RNPs, could not be reached with a single gene-editing approach. Taken together, this lipoMSN represents a versatile platform for the development of efficient, combinatorial gene-editing therapeutics.
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