FAM65B CRISPR/Cas9 KO Plasmid (h): sc-404100

Overview

FAM65B encodes a cytosolic protein implicated in regulation of small GTPase signaling, particularly pathways linked to Rho-family GTPases that coordinate actin cytoskeleton remodeling, cell polarity, and migration. Through these processes, FAM65B can influence immune cell activation dynamics and tissue-specific cellular motility programs, connecting it to broader signaling networks that shape adhesion and cytoskeletal organization. Altered expression or function of FAM65B has been explored in contexts where cytoskeletal control and immune signaling are perturbed, supporting its relevance for mechanistic studies in inflammation-associated phenotypes and cancer-related cell behavior. Functional interrogation of FAM65B therefore helps define how GTPase-regulated structural signaling interfaces with cellular state transitions.

FAM65B CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the FAM65B gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the FAM65B 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, FAM65B 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 FAM65B target site.
When co-transfected with FAM65B CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the FAM65B 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 FAM65B 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 FAM65B exon(s) critical for FAM65B 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.