CCRK CRISPR/Cas9 KO Plasmid (m): sc-430582

Overview

Cdk20 encodes cell cycle-related kinase (CCRK), a serine/threonine kinase implicated in regulation of cell-cycle progression and ciliary dynamics through kinase signaling networks. CCRK has been linked to activation of CDK family pathways, including modulation of CDK2 activity, and contributes to coordination of proliferation cues with centrosome and primary cilium-associated processes. In mouse models and mammalian cell systems, altered CCRK activity has been associated with changes in growth control and signaling pathway output, making it relevant to studies of oncogenic signaling and developmental regulation. These features position Cdk20/CCRK as a useful node for interrogating kinase-dependent mechanisms that couple cell-cycle control with tissue homeostasis.

CCRK CRISPR/Cas9 KO Plasmid (m) is a pool of plasmids designed for targeted disruption of the Cdk20 gene in mouse cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the Cdk20 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, CCRK HDR Plasmid (m) 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 Cdk20 target site.
When co-transfected with CCRK CRISPR/Cas9 KO Plasmid (m):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the Cdk20 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 Cdk20 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 Cdk20 exon(s) critical for CCRK 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.