QIAGEN Plasmid Plus 96 Miniprep Kit (4)

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Plasmids: For expression of Cas9 and sgRNAs from lentiviral vectors we optimized the sequence of pL-CRISPR.EFS.GFP (Addgene plasmid #57818, [39]), where the tracr sequence was modified to increase sgRNA stability and enhance its assembly with Cas9 protein [40]. Streptococcus pyogenes Cas9 and eGFP were linked via P2A and were expressed from the EFS promoter, whereas the sgRNAs were expressed from the human U6 pol III promoter. Protospacers, either targeting mutations, WT UTP14A or non-targeting, were cloned into pL-CRISPR.EFS.GFP by cloning complementary oligonucleotides into the vector. If necessary an additional G on the 5′-end was added. Likewise, protospacers were cloned into the pL.CRISPR.EFS.RFP vector, which was generated by replacing the GFP coding sequence with the RFP open reading frame. For the library preparation, oligos (Metabion, Planegg/Steinkirchen, Germany) were annealed, phosphorylated and ligated into pL-CRISPR.EFS.GFP using BsmBI sites. Ligations were transformed into E. coli DH5α and cells were grown 2× overnight in 2.0 ml DeepWell plates (thermofisher 278743) at 37 C with constant shaking. Plasmid DNA was first individually purified using QIAGEN Plasmid Plus 96 Miniprep Kit (Cat No: 960241), then equimolar ratios were pooled together, transformed and purified via Maxiprep (Qiagen, 16181) according to manufacturer’s protocol. The DNA was resuspended at 1 μg/μl and stored at − 20 °C.

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