Human Activity-Optimized CRISPR Knockout Library (3 sub-libraries in lentiCRISPRv1)
(Pooled Library #1000000100)

• Purpose:

  The Sabatini/Lander CRISPR pooled library is optimized for cleavage activity, in order to maximize the likelihood of gene knockout.

  The library is split into three sub-libraries. Two tubes each contain 90,000 gRNAs each or five sgRNAs per gene. The third tube contains 5,000 gRNAs.

• Vector Backbone:

  lentiCRISPR v1 (Plasmid #49535) - expresses Cas9

  Note: This plasmid has been discontinued, as an updated version is available. For confirmation of screen hits, Addgene encourages users to use the updated lentiCRISPR v2 (Plasmid #52961), which can be requested separately.

• Depositing Labs: David Sabatini, Eric Lander
• Publication: Wang et al Science. 2015 Oct 15. pii: aac7041.

Library Details

• Species: Human
• Genes targeted: 18,663
• gRNAs: 187,535
• Controls: 1,000 non-targeting, 500 intergenic
• Lentiviral Generation: 3rd

Library Shipping

Each library is delivered in microcentrifuge tubes on blue ice. A tube's contents will not necessarily be frozen. For best results, minimize freeze/thaws.

Pooled library #1000000100 will be delivered as three tubes, each containing a pooled sub-library. Note that each subpool must be transformed separately.

You will receive two sub-libraries with 90,000 gRNAs each at:

• Volume: ∼15 µL
• Concentration: 10 ng/µL

You will additionally receive one sub-library (hL3C9) with 5,000 gRNAs each at:

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

Resource Information

• Protocols:
  • Library Transformation Protocol (DOCX, 15 KB)
  • Library Screening Protocol (DOCX, 16 KB)
  • PCR Protocol for Illumina Sequencing Preparation (DOCX, 15 KB)
• Depositor Data:
  • List of Targeted Genes and sgRNA Sequences (XLSX, 10 MB)
  • Sequencing Data (sgRNA read counts for each subpool) (TXT, 2.9 MB)
  • Data for hL3C9 (XLSX, 11 MB)

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA

Industry Terms:

• Not Available to Industry

Trademarks:

• Zeocin® is an InvivoGen trademark.

Depositor Comments

The depositing lab notes that the DNA preps should be performed separately and then mixed together during virus production at ratios consistent with the pool size.

Figure 1. Diagram of Library Screening Process

When performing sgRNA validation in a Cas9-expressing cell line, the depositing laboratory recommends using plasmid pLenti-sgRNA (Addgene #71409).

Figure 2. sgRNA diversity after initial amplification

Figure 3. sgRNA diversity after infection of a target cell population