Pcr4blunt topo vector

The NKD2-specific guide RNA (forward 5′-CACCGACTCCAGTGCGATGTCTCGG -3′; reverse 5′-AAACCCGAGACATCGCACTGGAGTC -3′) were cloned into pL-CRISPR.EFS.GFP (Addgene #57818) using BsmBI restriction digestion. Lentiviral particles were produced by transient co transfection of HEK293T cells with lentiviral transfer plasmid, packaging plasmid psPAX2 (Addgene #12260) and VSVG packaging plasmid pMD2.G (Addgene #12259) using TransIT-LT (Mirus). Viral supernatants were collected 48-72 hours after transfection, clarified by centrifugation, supplemented with 10% FCS and Polybrene (Sigma-Aldrich, final concentration of 8μg/ml) and 0.45μm filtered (Millipore; SLHP033RS). Cell transduction was performed by incubating the PDGFRß cells with viral supernatants for 48hrs. eGFP expressing cells were single cell sorted into 96-well plates. Expanded colonies were assessed for mutations with mismatch detection assay: gDNA spanning the CRISPR target site was PCR amplified and analyzed by T7EI digest (T7 Endonuclease, NEB M0302S). To determine specific mutation events on both alleles within the clones grown, the PCR product was subcloned into the pCR™ 4Blunt-TOPO vector (Thermo Scientific K287520). Minimum 6 colonies per CRISPR-clone were grown and sent for sanger sequencing (Clone C2: 30 colonies have been sequenced).

Full-length DNA sequence of the S surface glycoprotein (SARS-CoV 2 isolate Wuhan-Hu-1, gene ID: 43740568, NC_045512.2:21563-25384) was used to design a plasmid for in vitro transcription (IVT). Briefly, a stretch of DNA sequence with two restriction sites, I-CeuI and I-SceI, was cloned into pCR4Blunt-TOPO vector (45-0031; Thermo Fisher Scientific). The full length of the S protein sequence was split to design two gBlocks (Integrated DNA Technologies, Coralville, Iowa, USA), each of which was approximately 1.5- and 2.3-kb long. The T7 promoter sequence (5’-taatacgactcactataggg-3’) was added to the 5’ end of the 1.5-kb gBlock. The two gBlocks were ligated into the backbone vector using I-CeuI, BstEII, and I-SceI. Following sequence confirmation by Sanger sequencing (Genewiz, South Plainfield, NJ, USA), the full-length S protein DNA sequence, cut by I-CeuI and I-SceI, was extracted from TAE agarose gel. IVT was done following the manufacturer’s manual of the MEGAscript Kit (AM1333; Thermo Fisher Scientific). High-yield RNA was treated with DNase and cleaned up using the Monarch RNA cleanup kit (T2040L; New England Biolab, Ipswich, MA, USA). Size-confirmed pure IVT S protein mRNA was aliquoted and stored in -80°C until nanoparticle loading. S protein mRNA was loaded into LSC-Exo and Lipo nanoparticles via the electroporation method described above.