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Mouse CRISPR Knockout Pooled Library (Brie)
(Pooled Library #73632, #73633, #73633-LV, #73633-LVC)

  • Purpose

    The mouse CRISPR Brie lentiviral pooled libraries were designed using optimized metrics which combine improved on-target activity predictions (Rule Set 2), with an off-target score, the Cutting Frequency Determination (CFD).

  • Vector Backbone

    Available in either a 1 vector (lentiCRISPRv2 backbone) or 2 vector (lentiGuide-Puro backbone) system.

Ordering

1 vector system (lentiCRISPRv2)
This backbone encodes Cas9

Item Catalog # Description Quantity Price (USD)
Pooled Library 73632 gRNA pooled library in lentiCRISPRv2 1 $500 Add to Cart
Available to Academic and Nonprofits Only

2 vector system (lentiGuide-Puro)
A Cas9 plasmid is NOT included with this item and will have to be ordered separately. Can be used in conjunction with lentiCas9-Blast (Addgene #52962) or with cell lines already expressing Cas9.

Item Catalog # Description Quantity Price (USD)
Pooled Library 73633 gRNA pooled library in lentiGuide-Puro 1 $500 Add to Cart
Lentiviral Prep 73633-LV Virus (1.3×108 TU, titer ≥ 1×107 TU/mL)
and Pooled Library DNA More Information
1 $2950 Add to Cart
Concentrated Lentiviral Prep 73633-LVC Virus (1.3×108 TU, titer ≥ 2×108 TU/mL)
and Pooled Library DNA More Information
1 $3750 Add to Cart
Available to Academic and Nonprofits Only

Library Details

  • Species
    Mouse
  • Genes targeted
    19,674
  • gRNAs
    78,637
  • Controls
    1000
  • Lentiviral Generation
    3rd

Library Shipping

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

For the 2 plasmid system only: A Cas9 plasmid is NOT included with this library and will have to be ordered separately.

  • Volume
    ∼20µL
  • Concentration
    50ng/µL

Information for Lentiviral Prep (Catalog # 73633-LV)(Back to top )

Purpose

Ready-to-use lentiviral pooled library for CRISPR screening in mouse cells. Use on cells that are stably expressing Cas9 to make edits across 19,674 genes in the mouse genome.

Delivery

  • Volume 13 mL
  • Titer ≥1x10⁷ TU/mL
  • Pricing $2700 USD for 13 mL viral preparation + $250 USD for pooled library DNA.
  • Storage Store at -80℃. Thaw just before use and keep on ice.
  • Shipment Viral particles are shipped frozen on dry ice. Pooled Library DNA (10µL at 50ng/µL) will also be included in the shipment.

Viral Production & Use

  • Packaging Plasmids psPAX2 (plasmid #12260)
  • Envelope pMD2.G (plasmid #12259)
  • Buffer DMEM +10% FBS
  • Selectable Marker Puromycin

Biosafety

Requestor is responsible for compliance with their institution's biosafety regulations. Lentivirus is generally considered BSL-2. AAV is generally considered BSL-1, but may require BSL-2 handling depending on the insert. Biosafety Guide

Viral Quality Control

Pooled Library Representation:
  • To confirm library representation and distribution, we perform next-generation sequencing on the purified plasmid DNA pool that was used to generate lentivirus.

Titering Method:
  • Colony formation assay : A549 cells were transduced with serial dilutions of 73633-LV and treated with puromycin. Puromycin-resistant colonies were expanded for approximately 2 weeks, stained with crystal violet, and counted.

Visit our viral production page for more information.

Addgene Comments

Shipment specifications:

Pooled libraries are shipped on dry ice as 13 mL of virus at a titer of >1×107 TU/mL, for a total of at least 1.3×108 infectious units. The 13 mL volume is divided among three 4 mL aliquots and a 1 mL aliquot. Each viral service request also includes virus associated DNA, which is a sample of the purified plasmid DNA pool that was used to generate lentivirus.

How to use this virus:

We recommend using 1 mL of virus to determine optimal infection conditions before performing screening-scale infections. Detailed methods for determining optimal infection conditions, screening, and validating hits are described in the original publication. Before using this virus, Addgene strongly recommends reading the original publication (Doench et al., 2016).

A brief and partial description of how to use this virus:

  1. Start by optimizing the infection conditions in your cell line in order to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.5–1. Infect 2.5×106 suspension (or 3×106 adherent) cells in each well of a 12-well dish at different MOIs. Determine which condition yields a 30-50% infection efficiency, and note the infection efficiency for this condition.

  2. The Brie CRISPR knockout pooled library has 78,637 gRNAs. To achieve the recommended representation of ~400 cells per sgRNA, a total of ~3.15×107 infected cells are needed. Calculate the number of cells on which to perform the infection by considering the infection efficiency noted in the optimized condition. For example, if the infection efficiency is 50%, perform the infection on 6.30×107 cells to ultimately achieve 3.15×107 infected cells.

  3. Perform the screening-scale infection with the pre-determined MOI in the same 12-well format as the optimized condition. Briefly, infect 2.5×106 suspension (or 3×106 adherent) cells in each well of a 12-well dish. Scale up the number of wells to achieve the desired total number of cells.

  4. Based on a screening MOI of 0.5-1 and the total number of cells infected, Addgene estimates that 3-10 mL of virus will be needed to perform the screen. An additional 1 mL of virus will be needed to perform the optimization. Based on the infection efficiency observed in each cell line, these estimates are subject to variation. Not every cell type is infectable under these conditions, and Addgene recommends that infectability of cells be determined empirically.

How to use the virus associated DNA:

Distribution of this pooled lentiviral library includes virus associated DNA, which is a sample of the purified plasmid DNA pool that was used to generate lentivirus.

  • This purified plasmid DNA pool can be sequenced and used as the starting plasmid DNA (pDNA) pool reference when performing screen analysis. As described in the original publication (Doench et al., 2016), perform screen analysis by determining the log2-fold-change of each single-guide RNA relative to the pDNA pool. The pDNA has been shown to serve as a good surrogate for an early time point and is more cost effective when performing many screens (Shalem et al., 2014).

  • The purified plasmid DNA pool can be amplified to make more pooled plasmid library stock DNA. See our Library Amplification Protocol for more information.

Information for Concentrated Lentivirus (Catalog # 73633-LVC) ( Back to top )

Purpose

Ready-to-use concentrated lentiviral pooled library for CRISPR screening in mouse cells. Use on cells that are stably expressing Cas9 to make edits across 19,674 genes in the mouse genome.

In addition to the viral particles, you will also receive purified Mouse sgRNA library Brie in lentiGuide-Puro plasmid DNA.

Delivery

  • Volume Depending on titer, volume will supply a minimum of 1.3×10⁸ transducing units.
  • Titer ≥2×10⁸ TU/mL
  • Pricing $3500 USD for viral preparation + $250 USD for pooled library DNA.
  • Storage Store at -80℃. Thaw just before use and keep on ice.
  • Shipment Viral particles are shipped frozen on dry ice. Pooled Library DNA (10µL at 50ng/µL) will also be included in the shipment.

Viral Production & Use

  • Packaging Plasmids psPAX2 (plasmid #12260)
  • Envelope pMD2.G (plasmid #12259)
  • Buffer PBS
  • Selectable Marker Puromycin
  • Purification Lentivirus was harvested from cell culture medium (DMEM + 10% FBS). Lentiviral particles were then collected by precipitation in polyethylene glycol (PEG) followed by centrifugation. Precipitated pellets containing viral particles were resuspended in PBS.

Biosafety

Requestor is responsible for compliance with their institution's biosafety regulations. Lentivirus is generally considered BSL-2. AAV is generally considered BSL-1, but may require BSL-2 handling depending on the insert. Biosafety Guide

Viral Quality Control

Titering Method:
  • Colony formation assay : A549 cells were transduced with serial dilutions of 73633-LVC and treated with puromycin. Puromycin-resistant colonies were expanded for approximately 2 weeks, stained with crystal violet, and counted. Read our colony formation titering assay protocol here.
Notes:
  • Quality control was performed on this lentiviral preparation prior to concentration. Results and explanations of quality control methods can be seen in the Information for Lentiviral Prep section on this page.

Visit our viral production page for more information.

Addgene Comments

Shipment specifications:

This concentrated pooled library is shipped on dry ice as a total of 1.3×108 transducing units. This library is provided at a minimum titer of 2×108 TU/mL. The total volume provided will depend on the titer and will contain the total number of specified transducing units. Each viral service request also includes virus associated DNA, which is a sample of the purified plasmid DNA pool that was used to generate lentivirus.

How to use this virus:
  • This is a concentrated viral preparation with titer ≥2×10⁸ TU/mL. For the same number of infections particles in a larger volume, see the standard Lentiviral Prep of this pooled library.

  • If using this virus for the first time on a particular cell line, we recommend determining optimal infection conditions before performing screening-scale infections. Detailed methods for determining optimal infection conditions, screening, and validating hits are described in the original publication. Before using this virus, Addgene strongly recommends reading the original publication (Doench et al., 2016).

  • A brief and partial description of how to use this virus can be found in the Information for Lentiviral Prepsection on this page.

How to use the virus associated DNA:

Distribution of this pooled lentiviral library includes virus associated DNA, which is a sample of the purified plasmid DNA pool that was used to generate lentivirus.

How to cite this pooled library ( Back to top )

These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the pooled libraries were described, and include Addgene in the Materials and Methods of your future publications.

  • Example for your Materials & Methods section:

    Mouse Brie CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73633)
  • For your References section:

    Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE. Nat Biotechnol. 2016 Jan 18. doi: 10.1038/nbt.3437. 10.1038/nbt.3437 PubMed 26780180