h-prune CRISPR/Cas9 KO Plasmid (h): sc-412914

Overview

PRUNE1 encodes h-prune, a member of the DHH phosphoesterase family with cyclic nucleotide phosphodiesterase and exopolyphosphatase activities that influence intracellular signaling and metabolic homeostasis. h-prune has been linked to regulation of cell motility and cytoskeletal dynamics through interactions that impact Rho family GTPase pathways and focal adhesion–associated processes. Altered PRUNE1 function is associated with neurodevelopmental phenotypes and has been investigated in contexts of aberrant proliferation and migration, making it relevant for dissecting mechanisms of cellular plasticity. In cultured systems, PRUNE1 perturbation is used to study how phosphoesterase-mediated signaling interfaces with nucleotide metabolism, stress responses, and migration programs.

h-prune CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the PRUNE1 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the PRUNE1 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, h-prune 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 PRUNE1 target site.
When co-transfected with h-prune CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the PRUNE1 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 PRUNE1 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 PRUNE1 exon(s) critical for h-prune 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.