Mouse Kinome CRISPR Knockout Library (Brie)
(Pooled Libraries #75316, #75317)
PurposeMouse kinome CRISPR knockout pooled libraries. Each library contains 2,852 unique sgRNAs targeting 713 mouse kinase genes for 4 guides per target.
Each library is delivered in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze-thaws.
The amplification protocol provided, which recommends using 400ng of DNA per transformation, was written in reference to the whole genome-wide libraries. These kinome-specific sub-pools contain fewer plasmids and thus require less DNA to achieve complete representation. Therefore, using the 100ng of DNA as provided for transformation is sufficient.
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.
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 Kinome CRISPR pooled library (Brie) was a gift from John Doench & David Root (Addgene # )
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