AY27_pU6.gRNA.CLYBL
(Plasmid #199238)

• Purpose: Expression construct encoding a S. pyogenes guide RNA targeting the human CLYBL safe harbor locus
• Depositing Lab: Manuel Goncalves
• Publication: Wang et al Nucleic Acids Res. 2023 Mar 17:gkad165. doi: 10.1093/nar/gkad165.

Backbone

• Vector backbone: pBR322
• Total vector size (bp): 3047
• Vector type: Mammalian Expression, CRISPR, Synthetic Biology

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: S. pyogenes gRNA spacer
• Species: H. sapiens (human), Synthetic
• Insert Size (bp): 20
• Promoter: RNA polymerase III promoter for human U6 snRNA

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BveI (destroyed during cloning)
• 3′ cloning site: BveI (destroyed during cloning)
• 5′ sequencing primer: M13 rev
• 3′ sequencing primer: M13 fwd

Resource Information

• Supplemental Documents:
  • AY27_pU6.gRNA.CLYBL.gbk

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This gRNA expression construct can be combined with target site-containing donor plasmids AD59_pEP.DonorCLYBL.TS (Addgene plasmid #199227) or AZ64_pE.DonorCLYBL.TS (Addgene plasmid #199228) to insert foreign DNA at the human CLYBL safe harbor locus using SpCas9 nucleases (HMEJ-based gene targeting) or SpCas9.D10A nickases (ITPN-based gene targeting).

Note: to reduce the chances for off-targeting donor DNA insertions when applying HMEJ-based genome editing it is preferable using AW01_pU.CAG.eSpCas9(1.1).rBGpA (Addgene plasmid #199255). Combining this reagent with a S. pyogenes Cas9 nickase (e.g., Addgene plasmids #199256, #199253, #199254 or #199252) leads to seamless ITPN-mediated gene targeting.