BARBEKO sgRNA Library
(Pooled Library #174163)

• Purpose:

  BARBEKO, short for iBARed cytosine Base Editing-mediated gene KnockOut, leverages CRISPR cytosine base editors for genome-scale knockout screens in human cells by perturbing gene start codons, splice sites, or by introducing premature termination codons. It is integrated with iBAR, a design in the sgRNA scaffold for improving screening quality and efficiency.

  Each sgRNA construct contains one iBAR sequence and each sgRNA is followed by four iBARs (214,008 sgRNA^iBARs in total).

• Vector Backbone:

  pLenti-sgRNA-Lib - does not express Cas9

  This library is designed for gene knockout screens by using CRISPR cytosine base editors (CBE), but not Cas9. Library was re-constructed into lentiviral sgRNA-iBAR backbone according to the method in Zhu et al., 2019 PMID: 30678704.

• Depositing Labs: Wensheng Wei
• Publication: Xu et al Nat Biotechnol. 2021 Jun 21. pii: 10.1038/s41587-021-00944-1. doi: 10.1038/s41587-021-00944-1.

Library Details

• Species: Human
• Genes targeted: 17,501
• gRNAs: 53,502
• Controls:

  
  Non-targeting controls: 500
  Negative controls targeting safe-harbor regions: 499

• 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 Amplification Protocol (21.6 KB)
  • PCR Protocol for NGS Analysis (22.1 KB)
• Depositor Data:
  • Library information and list of targeted genes (1.9 MB)
  • sgRNA-iBAR counts (5.3 MB)
  • Count sgRNAs with barcode from paired-end fastq files (3.7 MB)
• Terms and Licenses:
  • UBMTA

Depositor Comments

• CBE with sgRNAs targeting start codons, splice acceptor sites, splice donor sites, and codons of glutamine, arginine or tryptophan disrupting gene functions.

The CBE-expressing cassette from pCMV_AncBE4max (Addgene #112094) was transferrred into a lentiviral backbone to create pLenti-AncBE4max (Addgene #174164). To efficiently package the CBE into lentivirus, concentrate the lentiviral supernatants before use.

For cell fitness screens, ZFCiBAR algorithm can be used and obtained from https://github.com/wolfsonliu/zfc. For positive selection screens, MAGeCK^iBAR algorithm can be used for analysis (Zhu et al., 2019): https://bitbucket.org/WeiLab/mageck-ibar/.