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Objective(s): Colorectal cancer (CRC) is a major cause of mortality, and KRAS mutations drive tumorigenesis and therapy resistance. Although CRISPR-Cas9 enables precise genome editing, intracellular delivery of Cas9 ribonucleoproteins (RNPs) remains challenging. We evaluated direct electroporation of Cas9-sgRNA RNPs targeting KRAS p.G12V in SW480 CRC cells to assess delivery efficiency, on-target editing, off-target activity, and viability effects. Materials and Methods: An sgRNA targeting KRAS p.G12V was designed computationally. Cas9-sgRNA RNPs were assembled and validated by in vitro digestion. RNPs were electroporated into SW480 cells using a square-wave electroporation protocol; pmaxGFP served as the delivery control. On-target editing and five predicted off-target loci were assessed by Sanger sequencing and TIDE analysis. Cell viability was measured by the MTT assay.Results: Electroporation resulted in high GFP expression (~97%). TIDE analysis at the KRAS locus estimated ~27% tot

High-throughput loading of sgRNA:Cas9 RNPs targeting Myo7aSh1 into extracellular vesicles prevents progressive hearing loss in an adult mouse model.

Eukaryotic genomes generate a plethora of polyadenylated (pA+) RNAs1,2, which are packaged into ribonucleoprotein particles (RNPs). To ensure faithful gene expression, functional pA+ RNPs, including protein-coding RNPs, are exported to the cytoplasm, whereas transcripts within non-functional pA+ RNPs are degraded in the nucleus1–4. How cells distinguish these opposing fates remains unknown. The DExD-box ATPase UAP56 (also known as DDX39B) is a central component of functional pA+ RNPs, and promotes their docking to the nuclear pore complex-anchored TREX-25,6, which triggers transcript release from UAP56 to facilitate export7. Here we reveal that the poly(A) tail exosome targeting (PAXT) connection8 binds a TREX-2-like module, which releases pA+ RNAs from UAP56 for decay by the nuclear exosome. The core of this module consists of a LENG8–PCID2–SEM1 trimer, which we show is structurally and biochemically equivalent to the central GANP–PCID2–SEM1 trimer of TREX-2. Mutagenesis and transcrip

High-throughput loading of sgRNA:Cas9 RNPs targeting Myo7aSh1 into extracellular vesicles prevents progressive hearing loss in an adult mouse model.

The regulation of gene expression requires many RNA-protein complexes, or ribonucleoproteins (RNPs). Understanding the molecular mechanisms guiding th…

Abstract. Effective genome editing requires a sufficient dose of CRISPR–Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune res

Eukaryotic genomes generate a plethora of polyadenylated (pA+) RNAs, that are packaged into ribonucleoprotein particles (RNPs). To ensure faithful gene expression, functional pA+ RNPs, including prote...

Track 1 / 9 (Complete Soundtrack) System: Nintendo Entertainment System Developer: Software Creations Publisher: Arcadia Systems (US) Composers: Tim Follin, Geoff Follin Year: 1990 Tracklist: 1 - Title Screen 2 - Summoned by Galactus 3 - World Selected 4 - BGM I 5 - BGM II 6 - Section Completed 7 - Device Completed 8 - Game Over 9 - Hi Score Download this soundtrack- 224k MP3: http://tinyurl.com/yacmgyc NSF playback sampled from nsfplay.