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Adapting prime editing with split prime editors in Escherichia coli and its application to Staphylococcus aureus genome editing.
Prime editing is a precise and rapid genome-editing technique that modifies short DNA sequences using tailored guide RNAs. To implement this technique in bacteria, we used Prime Editor 2 (PE2) with the DeepPrime gRNA design tool and assessed its gene-editing efficiency in Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) cells. Our findings indicate that a split PE2, comprising a reverse transcriptase and two Cas9 nickase domains, exhibited gene-editing efficiency comparable to that of the intact PE2. The efficiency observed in E. coli was significantly affected by the target sites, edit type, and the presence of exonucleases. In MRSA, which serves as a model to evaluate the applicability in non-model bacterial species, Streptococcus pyogenes PE2 (SpPE2) exhibited superior performance relative to Staphylococcus aureus PE2 (SaPE2). Furthermore, the split SpPE2 lacking the reverse transcriptase successfully induced the intended mutation in MRSA. This study demonstrates the feasibility of prime editing within bacterial systems.
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Introducing Emerging Science for the Clinician.
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Technologies and Computational Analysis Strategies for CRISPR Applications.
The CRISPR-Cas system offers a programmable platform for eukaryotic genome and epigenome editing. The ability to perform targeted genetic and epigenetic perturbations enables researchers to perform a variety of tasks, ranging from investigating questions in basic biology to potentially developing novel therapeutics for the treatment of disease. While CRISPR systems have been engineered to target DNA and RNA with increased precision, efficiency, and flexibility, assays to identify off-target editing are becoming more comprehensive and sensitive. Furthermore, techniques to perform high-throughput genome and epigenome editing can be paired with a variety of readouts and are uncovering important cellular functions and mechanisms. These technological advances drive and are driven by accompanying computational approaches. Here, we briefly present available CRISPR technologies and review key computational advances and considerations for various CRISPR applications. In particular, we focus on the analysis of on- and off-target editing and CRISPR pooled screen data.
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Generation of a new DiCre expressing parasite strain for functional characterization of Plasmodium falciparum genes in blood stages.
Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria.
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Genetically modified humans: the X-Men of scientific research.
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Muscle satellite cell editing by LNP-CRISPR-Cas9 to resist muscle injury.
Muscle satellite cells are essential for skeletal muscle regeneration and represent an attractive therapeutic target for gene delivery in Duchenne muscular dystrophy (DMD). However, efficient in vivo transduction of these cells has remained challenging. Here, we demonstrate that lipid nanoparticle (LNP)-mediated delivery of Streptococcus pyogenes CRISPR-Cas9 mRNA and guide RNA (LNP-CRISPR) induces exon skipping in Pax7-positive satellite cells more efficiently than adeno-associated virus (AAV) vectors following intramuscular or intravenous administration in a DMD mouse model. Furthermore, unlike AAV-CRISPR, LNP-CRISPR-mediated genome editing showed greater resistance to repeated muscle injuries, indicating successful editing of regenerative satellite cells. These results highlight the potential of LNPs as a non-viral platform for durable genome editing in skeletal muscle and lay the foundation for developing safe and sustainable genome-editing therapies for DMD.
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CRISPR/Cas9 and p53: An Odd Couple Requiring Relationship Management.
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Prime editing GEMMs to model cancer mutations.
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A toolkit for microbial community editing.
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TO EDIT OR NOT: THE NGAGO STORY.
New genome-editing approaches always receive widespread attention. But in the case of a novel Argonaute-based technique published last spring, attention has been particularly intense.
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