Turner Lab Human messenger-RBP sgRNA Library
(Pooled Library #168791)

• Purpose:

  This lentiviral CRISPR knockout library targets human messenger-RNA binding proteins with 10 single guide RNAs (sgRNAs) per gene. The lentiviral backbone encodes both puromycin resistance and the CD90.1 selection marker.

• Vector Backbone:

  • pKLV2_hU6_BbsI_ccdB_BbsI_iScaffold_mPGK_puro_T2A_Thy1.1
    • Does not express Cas
    • Not available through Addgene
  • Plasmid annotation and sequence (Genbank) (12.7 KB)
  • Plasmid description: PKLV2_hU6_BbsI-ccdB-BbsI_iScaffold_mPGK_puro-2A-Thy1.1 (PKLV2_sgRNA_puro-Thy1.1) was generated in two rounds of cloning from Addgene Plasmid 67974: pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W. Firstly, a PCR product encoding Thy1.1 and the appropriate flanking sequences was generated and ligated (Gibson assembly) into a XhoI + NotI linearised vector (Addgene Plasmid 67974). Secondly, a PCR product encoding the ccdB toxic gene, a region validated to contain the ccdB gene’s uncharacterised promoter, and the appropriate flanking sequences were generated and then ligated (Gibson assembly) into a BbsI linearised intermediate vector (PKLV2_hU6_BbsI-BbsI_iScaffold_mPGK_puro-2A-Thy1.1).

• Depositing Labs: Martin Turner
• Publication: Turner et al CRISPR J. 2021 Jun;4(3):427-437. doi: 10.1089/crispr.2020.0116. Epub 2021 Jun 4.

Library Details

• Species: Human
• Genes targeted: 725
• gRNAs: 8,260
• Controls: 500 non-targeting sgRNAs
• Lentiviral Generation: 3rd

Library Shipping

This library is delivered as suspended DNA in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze-thaws.

• Volume: ∼20µL
• Concentration: 50ng/µL

Resource Information

• Protocols:
  • Library Transformation and Sequencing Protocols (20.5 KB)
• Depositor Data:
  • Library Design and Representation Overview (160.5 KB)
  • NGS Primer Table (9.4 KB)
  • Table of gene targets, gRNA sequences, and count data (243.7 KB)
• Terms and Licenses:
  • UBMTA