Clone Assay:Article Title: Genome writing and Targeted Delivery of the NKX6-3/ANK1 gene cluster and its Type 2 Diabetes GWAS Variants to Human iPSCs
Article Snippet: Oligos orNCH001 and orNCH002 were annealed to form the left-side gRNA-targeting upstream of endogenous NKX6-3 . .. Oligos orNCH003 and oNCH004 were annealed to produce the right-side cutting downstream of sANK1 . gRNAs targeting MC1 for payload deliveries were cloned into pX333 (Addgene: 64073), which contains two independent gRNA expression cassettes followed by a Cas9 cassette. .. Oligos orNCH009 and oNCH010 were annealed to form the gRNA targeting the left side of MC1 and outside the left homology arms of the synthetic payload, and orNCH011 and orNCH012 were annealed to target the right side of MC1 and outside the right homology arms of synthetic payload using Golden Gate Assembly in a two-step process using BbsI (NEB: R3539S) and BsaI (NEB: R3733S) ( ).
Article Title: A GABAergic network from AVP- to VIP-neurons in the suprachiasmatic nucleus sets the timing of circadian behavior rhythms.
Article Snippet: .. Oligonucleotides encoding the guide sequences were cloned into the BbsI and BsaI sites of pX333 (Addgene #64073, a gift from Dr. Andrea Ventura) [50]. .. Then, a fragment containing two tandem units of U6-gRNA was amplified by PCR, using the following primers: 5′-agtacgcgTCGAGCATGCTCGAGAATGG-3′ and 5′-agtacgcgtCGGGTAC CCCATTTGTCTGC-3′, and cloned into the MluI site of pAAV-EF1a-DIO-mCherry (a gift from Dr. Bryan Roth) as described previously [48]. pAAV-U6-gGabrb2-EF1α-DIO-mCherry happened to contain two copies of the amplified fragment. pAAVU6-gControl-EF1α-DIO-mCherry contains spacer sequences from pX333 instead of gRNA sequences for Gabrb genes. pAAV-CAG-FLEX-ChrimsonR-mCherry was described previously [12].
Article Title: Neuronal feedback loop of the suprachiasmatic nucleus generates robust circadian rhythms.
Article Snippet: .. Oligonucleotides encoding the guide sequences were cloned into the BbsI and BsaI sites of pX333 (Addgene #64073, a gift from Dr. Andrea Ventura)74. .. Then, a fragment containing two tandem units of U6-gRNA was amplified by PCR, using the following primers: 5’-agtacgcgTCGAGCATGCTCGAGAATGG-3’ and 5’- agtacgcgtCGGGTACCCCATTTGTCTGC-3’, and cloned into the MluI site of pAAV-EF1aDIO-mCherry (a gift from Dr. Bryan Roth) as described previously75. pAAV-U6-gControl-EF1αDIO-mCherry contains spacer sequences from pX333 instead of gRNA sequences for Vipr2. pAAV-EF1α-DIO-GFP::TeNT was kindly provided by Dr. Richard D. Palmiter76.
Article Title: A GABAergic network from AVP- to VIP-neurons in the suprachiasmatic nucleus sets the timing of circadian behavior rhythms
Article Snippet: .. Oligonucleotides encoding the guide sequences were cloned into the BbsI and BsaI sites of pX333 (Addgene #64073, a gift from Dr. Andrea Ventura) [ ]. .. Then, a fragment containing two tandem units of U6-gRNA was amplified by PCR, using the following primers: 5′-agtacgcgTCGAGCATGCTCGAGAATGG-3′ and 5′-agtacgcgtCGGGTACCCCATTTGTCTGC-3′, and cloned into the MluI site of pAAV-EF1a-DIO-mCherry (a gift from Dr. Bryan Roth) as described previously [ ]. pAAV-U6-gGabrb2-EF1 α- DIO-mCherry happened to contain two copies of the amplified fragment. pAAV-U6-gControl-EF1 α- DIO-mCherry contains spacer sequences from pX333 instead of gRNA sequences for Gabrb genes. pAAV - CAG-FLEX-ChrimsonR-mCherry was described previously [ ].
Article Title: Neuronal feedback loop of the suprachiasmatic nucleus generates robust circadian rhythms
Article Snippet: .. Oligonucleotides encoding the guide sequences were cloned into the BbsI and BsaI sites of pX333 (Addgene #64073, a gift from Dr. Andrea Ventura) . .. Then, a fragment containing two tandem units of U6-gRNA was amplified by PCR, using the following primers: 5′-agtacgcgTCGAGCATGCTCGAGAATGG-3′ and 5′-agtacgcgtCGGGTACCCCATTTGTCTGC-3′, and cloned into the MluI site of pAAV-EF1a-DIO-mCherry (a gift from Dr. Bryan Roth) as described previously . pAAV-U6-gControl-EF1α-DIO-mCherry contains spacer sequences from pX333 instead of gRNA sequences for Vipr2 . pAAV-EF1α-DIO-GFP::TeNT was kindly provided by Dr. Richard D. Palmiter .
Expressing:Article Title: Genome writing and Targeted Delivery of the NKX6-3/ANK1 gene cluster and its Type 2 Diabetes GWAS Variants to Human iPSCs
Article Snippet: Oligos orNCH001 and orNCH002 were annealed to form the left-side gRNA-targeting upstream of endogenous NKX6-3 . .. Oligos orNCH003 and oNCH004 were annealed to produce the right-side cutting downstream of sANK1 . gRNAs targeting MC1 for payload deliveries were cloned into pX333 (Addgene: 64073), which contains two independent gRNA expression cassettes followed by a Cas9 cassette. .. Oligos orNCH009 and oNCH010 were annealed to form the gRNA targeting the left side of MC1 and outside the left homology arms of the synthetic payload, and orNCH011 and orNCH012 were annealed to target the right side of MC1 and outside the right homology arms of synthetic payload using Golden Gate Assembly in a two-step process using BbsI (NEB: R3539S) and BsaI (NEB: R3733S) ( ).
Article Title: Improved vector toolkit for genome writing in mammalian cells
Article Snippet: .. To this end, we have used pX333 (Addgene plasmid # 64073) a vector for tandem expression of two sgRNAs from two independent U6 promoters . ..
Plasmid Preparation:Article Title: Improved vector toolkit for genome writing in mammalian cells
Article Snippet: .. To this end, we have used pX333 (Addgene plasmid # 64073) a vector for tandem expression of two sgRNAs from two independent U6 promoters . ..
Modification:Article Title: Composition and RNA binding specificity of metazoan RNase MRP
Article Snippet: .. Modified plasmids pCRIS-PITChv2-BSD-dTAG (Addgene, 91792), pCRIS-PITChv2-dTAG-BSD (Addgene, 91795), and pX333 (for simultaneous delivery of the PITCh and gene-targeting gRNAs; Addgene, 64073) were introduced into the cells via transfection. .. The HEK293T Rmp64-degradable cell line was generated via infection with lentivirus generated from the pLEX_305-C-dTAG plasmid (Addgene, 91798) to drive degradation tag (dTAG)-inducible expression of Rmp64, and modified pX333 (for delivery of RMP64 gene-targeting gRNAs; Addgene, 64073) were introduced into the cells via transfection to knockout the endogenous RMP64 gene.
Transfection:Article Title: Composition and RNA binding specificity of metazoan RNase MRP
Article Snippet: .. Modified plasmids pCRIS-PITChv2-BSD-dTAG (Addgene, 91792), pCRIS-PITChv2-dTAG-BSD (Addgene, 91795), and pX333 (for simultaneous delivery of the PITCh and gene-targeting gRNAs; Addgene, 64073) were introduced into the cells via transfection. .. The HEK293T Rmp64-degradable cell line was generated via infection with lentivirus generated from the pLEX_305-C-dTAG plasmid (Addgene, 91798) to drive degradation tag (dTAG)-inducible expression of Rmp64, and modified pX333 (for delivery of RMP64 gene-targeting gRNAs; Addgene, 64073) were introduced into the cells via transfection to knockout the endogenous RMP64 gene.
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