USP27X CRISPR/Cas9 KO Plasmid (h): sc-407628

Overview

USP27X encodes a deubiquitinating enzyme in the ubiquitin–proteasome system that removes ubiquitin chains from protein substrates to modulate their stability, localization, and signaling output. Through deubiquitination, USP27X contributes to regulation of proteostasis and cellular stress responses, influencing processes such as cell-cycle control, DNA damage signaling, and transcriptional programs. Altered ubiquitin signaling and dysregulated deubiquitination are recurrent features of cancer and neurodegenerative disease biology, making USP27X a useful node for pathway interrogation in these contexts. Functional studies of USP27X support mechanistic research into ubiquitin-dependent control of protein turnover and associated signaling networks.

USP27X CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the USP27X gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the USP27X locus, alongside the Streptococcus pyogenes Cas9 nuclease, and encodes GFP to enable fluorescent identification and enrichment of successfully transfected cells. This multi-guide strategy increases the likelihood of inducing frameshifts or deletions that produce a functional knockout, offering a more robust alternative to single-guide approaches. DSBs induced at multiple sites are resolved through non-homologous end joining (NHEJ) or, when used with the included HDR donor template, homology-directed repair (HDR) at a defined target site within the locus.

When used in conjunction with the RFP-expressing HDR donor, GFP and RFP fluorescence can be used together to distinguish transfected from edited cell populations, streamlining flow cytometry-based sorting and clone selection workflows.

Homology-Directed Repair (HDR) Donor — Puromycin Cassette with RFP Reporter

For applications requiring confirmed, selectable knockout clones, USP27X HDR Plasmid (h) includes an HDR donor construct containing a puromycin resistance cassette (PuroR) and a red fluorescent protein (RFP) reporter, flanked by homology arms specific to a defined USP27X target site.
When co-transfected with USP27X CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the USP27X open reading frame.
• RFP fluorescence provides an immediate visual indicator of successful integration, enabling fluorescence-based identification or sorting of edited cells prior to or alongside puromycin selection.
• Successfully edited cells are confirmed through puromycin resistance, substantially reducing clone screening burden.
• This selection strategy is ideal for generating stable, clonal KO cell lines for downstream functional studies, drug screening, or model development.

Cre-lox Cassette Removal System

The HDR donor construct features loxP sites flanking the PuroR-RFP selection cassette to allow clean marker removal following clone confirmation. Transient expression of Cre recombinase via the included Cre Vector: sc-418923 excises the cassette, leaving a minimal residual loxP site within the USP27X locus and eliminating potential confounding effects on downstream assays.
This two-step approach:

• Minimizes disruption to local chromatin architecture and neighboring regulatory elements
• Restores a near-native genomic context at the edited locus
• Enables reuse of the puromycin selection strategy in the same cell line for additional edits

Key Features

• gRNA targeting USP27X exon(s) critical for USP27X function
• Co-expression of SpCas9 and sgRNA from a single plasmid for simplified delivery
• HDR donor with puromycin resistance for positive clone selection
• loxP-flanked PuroR cassette with Cre recombinase vector for seamless marker removal
• Supplied ready to use for delivery by transfection

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.