FACE-1 CRISPR/Cas9 KO Plasmid (h): sc-403350

Overview

ZMPSTE24 encodes the zinc metalloprotease FACE-1, an endoplasmic reticulum membrane enzyme that catalyzes the final proteolytic processing step of prelamin A to mature lamin A. This maturation pathway is essential for nuclear lamina integrity, chromatin organization, and proper cell-cycle and stress responses, and it intersects with broader proteostasis and ER-associated processes through regulated membrane proteolysis. Loss of ZMPSTE24 activity causes toxic accumulation of farnesylated prelamin A, leading to abnormal nuclear morphology and altered transcriptional programs. Dysfunction in this pathway is linked to laminopathy biology, including premature aging syndromes and neuromuscular phenotypes, making FACE-1 a key node for studying nuclear envelope homeostasis.

FACE-1 CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the ZMPSTE24 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the ZMPSTE24 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, FACE-1 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 ZMPSTE24 target site.
When co-transfected with FACE-1 CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the ZMPSTE24 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 ZMPSTE24 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 ZMPSTE24 exon(s) critical for FACE-1 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.