Inzolia Human CRISPR/Cas12a Multiplex Knockout Pooled Library
(Pooled Library #209551, #209552)

• Purpose:

  The human, genome-wide, CRISPR knockout enAsCas12a gRNA library targets single genes and paralog pairs, triples, and quads with arrays of four gRNAs. 19,687 single genes are targeted with two constructs, each of which has four guides, with the second construct encoding the same gRNAs in different order in the array. 4,435 paralog pairs are targeted by a single construct with two gRNAs per gene. 376 paralog triples are targeted by a single construct with one gRNA per gene and are padded with a fourth non-targeting guide to extend the 3-mer to a 4-mer array. Finally, 100 paralog quads are targeted by a single construct with one gRNA per gene. The library also contains 20 arrays targeting EGFP, 500 arrays targeting intergenic sites, and 50 arrays targeting non-targeting sites for a total of 49,766 arrays in the library.

• Vector Backbone:

  pRDA_052 - does not express enAsCas12a

  pRDA_550 - expresses enAsCas12a

• Depositing Labs: John Doench
• Publication: Anvar et al. bioRxiv. 2023. doi: 10.1101/2023.01.03.522655 .

Library Details

• Species: Human
• Genes targeted:
  
  • 19,687 single genes
  • 4,435 paralog pairs
  • 376 paralog triples
  • 100 paralog quads
• gRNAs:
  
  • for single genes: 4 guides/gene/construct x 2 constructs (second construct has the same 4 guides in a different order in the array)
  • for paralog pairs: 2 guides/gene x 2 genes/construct
  • for paralog triples: 1 guide/gene x 3 genes/construct (non-targeting sequences added to extend 3mer paralog constructs into 4mer guide arrays)
  • for paralog quads: 1 guide/gene x 4 genes/construct
• Controls:
  
  • 20 arrays targeting EGFP
  • 500 arrays targeting intergenic site
  • 50 arrays targeting non-targeting sites
• Total number of arrays: 49,766
• 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: ∼25 µL
• Concentration: 50 ng/µL

Resource Information

• Protocols:
  • Inzolia Library Amplification Protocol (PDF, 175 KB)
  • Sequencing Protocol (PDF, 311 KB)
• Depositor Data:
  • Inzolia Library - Annotations (CSV, 15 MB)
  • Inzolia Library in pRDA_052 - Read Counts (ZIP, 3.1 MB)
  • Inzolia Library in pRDA_550 - Read Counts (ZIP, 3.1 MB)
• Terms and Licenses:
  • UBMTA

Depositor Comments

• In4mer platform for whole-genome screening. Inzolia human whole-genome library targets single genes and paralog pairs, triples, and quads with arrays of four Cas12a gRNAs. Each gene or gene family is targeted by two arrays. Commercially synthesized oligo pools were cloned into the one component pRDA_052 or pRDA_550 lentiviral vector; schematic created in Biorender.