Mouse CRISPR Knockout Pooled Library (Brie)
(Pooled Library #73632, #73633, #73633-LV)
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).
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.
Please see Sanson et al. Nat Commun. (2018) for additional information.
NOTE: Lentiviral vectors can recombine during library amplification, resulting in an ~1kb band containing the origin and ampicillin resistance cassette. This recombination product should not adversely affect library function as it does not contain lentiviral packaging sequences or the PuroR gene and will not be selected during screening. If this 1kb band is observed after amplification, we recommend starting with a larger prep (gigaprep) from the original sample or gel purifying the amplified library to remove the recombinant band and performing another transformation with the purified sample.
Information for Lentiviral Prep (Catalog # 73633-LV)(Back to top )
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.
- Volume Varies depending on titer.
- Titer ≥5 x 10⁶ TU/mL
- Pricing $1700 USD 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
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
To confirm library representation and distribution, we perform next-generation sequencing on the purified plasmid DNA pool that was used to generate lentivirus.
- ddPCR: 293T cells are transduced with serial dilutions of 73633-LV, harvested several days later, and genomic DNA is isolated. Copies of RRE are measured and normalized to RPP30.
- PCR was carried out with primers targeting the U6 and EF1-alpha promoters. The PCR product was visualized on an agarose gel for size confirmation.
- Forward primer: GAGGGCCTATTTCCCATGATT
- Reverse Primer: CACGGCGACTACTGCACTTA
Visit our viral production page for more information.
Addgene CommentsShipment specifications:
Pooled libraries containing at least 1.25×108 infectious units are shipped on dry ice at a titer of ≥5×106 TU/mL. The total volume is divided among several large aliquots and one 1.3 mL aliquot for testing. 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:
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.
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.
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.
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.
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.
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. PubMed 26780180