- Description PEmax Enzyme Also Available Intact Genomics is your resource for in vitro gene editing using CRISPR Cas systems. Prime-editing is gaining popularity as a precision gene-editing technique. Prime-editing (PE) enzymes are a fusion of the Cas9 nickase mutant (H840A) with a modified Murine Moloney Leukemia Virus-Reverse Transcriptase (MMLV-RT) (1,2). One of the resulting optimized prime-editing fusions is known as PEmax enzyme (3). Prime-editing enzymes enable precise gene-editing without the need for double-stranded DNA breaks (DSBs) or donor DNA templates. PE enzymes require a unique long gRNA known as a “pegRNA” shown schematically in Fig.1. We understand your need to generate sufficient quantities of pegRNA for in vitro gene editing projects. The IG® PE pegRNA synthesis kit is simple to use, and is scalable to help you reach necessary yields of pegRNA. The kit can be purchased with an optional IG® RNA Cleanup Kit (Cat.#4003 or #4005) for purification of pegRNA. The process of pegRNA synthesis by in vitro transcription (IVT) is intricate but allows for successful creation of longer pegRNAs (e.g. 100 nt or more) that cannot be easily chemically synthesized and purified at a low cost. pegRNA synthesis is often complicated, and the maximum yield of pegRNA depends on the edit region. Only Intact Genomics provides an IVT pegRNA synthesis kit for PE enzymes. Our kit provides at least 7ug of pegRNA per 20 µL reaction, enough for in vitro DNA sequence editing in most cases. This kit also provides a protocol for an optional 100 µL pegRNA synthesis volume to achieve more yield. Higher yields from the IG® PE pegRNA synthesis kit will enhance your chances of experimental success. Intact Genomics is with you every step of the way to complete your projects. We offer expert advice, fast ordering and delivery, and product customization. For successful pegRNA synthesis, a scientist on your team must identify a DNA sequence to edit and order unique primers targeting that sequence. (See oligo design instructions in this manual.) From there, a simple workflow details the in vitro pegRNA synthesis steps, and you may progress to prime-editing using PE enzyme, optionally, IG® PEmax enzyme (Cat.#3473 or #3476). Benefits The IG® PE pegRNA Synthesis Kit is a perfect choice for a variety of pegRNA synthesis needs. Below is a sampling of some of the key benefits. IG® PE pegRNA Synthesis Kit includes everything but your target oligos (all buffers, enzymes, scaffolds). User simply needs to provide the oligos for your target: a pair of sgRNA oligo and pegRTpbs oligo generated using the template design in this manual. Rapid Workflow (Less than 1 hour) Scalable Yield (100 µl reaction) Customer support – our team is available to aid with your success. Intact Genomics is with you every step of the way to complete your projects. We offer expert advice, fast ordering and delivery , and product customization. Components and Storage IG® PE pegRNA Synthesis Kit contains the items below. Store all components at -20°C. IG® PE pegRNA Enzyme Mix (10X) IG® PE pegRNA Reaction Mix. (4X) igDNase I (RNase-free, 2 Units/µL) IG® PE pegRNA Control Oligo (1 µM) Dithiothreitol (DTT, 0.2 M) NTPs mix (25 mM) Items needed but not provided in this kit: Nuclease-Free Water Custom nucleotide oligos (see instructions below to design your oligos) Nuclease-free pipet tips and microcentrifuge tubes References: Anzalone AV, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576:149–157. Lee J, Lim K, Kim A, et. al. Prime editing with genuine Cas9 nickases minimizes unwanted indels. Nat Commun. 2023 Mar 30;14(1):1786. Chen PJ, Hussmann JA, Yan J, et. al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 2021 Oct 28;184(22):5635-5652.e29. 3404 3406
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