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For sgRNA cloning into the PX330 (PX330-Nanog_sgRNA1, PX330-Nanog_sgRNA2, PX330-Esrrb_sgRNA1, PX330-Esrrb_sgRNA2, Additional file 9: Table S8, [88]) or PX458 plasmid (PX458-Dusp9_sgRNA1, [89]), two complementary oligos containing the guide sequence and a BbsI recognition site (Oligo F: 5′CACCGNNNNNNNNNN … .3′ and Oligo R: 5′AAACNNNNNNNNNN … ..C3´) were annealed and cloned into the BbsI (NEB) digested target plasmid.

sgRNAs for CRISPRa (pU6-Dusp9.1-EF1Alpha-puro-T2A-BFP, pU6-Dusp9.2-EF1Alpha-puro-T2A-BFP, pU6-Klhl13.1-EF1Alpha-puro-T2A-BFP, pU6-Klhl13.2-EF1Alpha-puro-T2A-BFP, pU6-NTC.1-EF1Alpha-puro-T2A-BFP, pU6-NTC.2-EF1Alpha-puro-T2A-BFP) were cloned into a BlpI and BstXI digested pU6-sgRNA-EF1a-puro-T2A-BFP plasmid (Addgene 60955, [90]) by annealing oligos containing the guide sequence and recognition sites for BlpI and BstXI (Oligo F: 5′TTGGNNN...NNNGTTTAAGAGC3′ and Oligo R: 5′TTAGCTCTTAAACNNN...NNNCCAACAAG3′) and ligating them together with the linearized vector using the T4 DNA ligase enzyme (NEB).

For the CRISPRi validation of Dusp9 and Klhl13 (SP199_multi_Dusp9_CRISPRi and SP199_multi_Klhl13_CRISPRi, Additional file 9: Table S8), as well as the CRISPRi/a validation of putative Klhl13 interacting partners Alg13, Cct3, Larp1, Peg10, and Scml2 (SP199_multi_NTC1, SP199_multi_NTC2, SP199_multi_Alg13_CRISPRi, SP199_multi_Cct3_CRISPRi, SP199_multi_Larp1_CRISPRi, SP199_multi_Peg10_CRISPRi, SP199_multi_Alg13_CRISPRa, SP199_multi_Cct3_CRISPRa, SP199_multi_Larp1_CRISPRa, SP199_multi_Peg10_CRISPRa, SP199_multi_Peg10_CRISPRa_2 and SP199_multi_Scml2_CRISPRa, Additional file 9: Table S8), three different sgRNAs targeting each gene were cloned into a single sgRNA expression plasmid with Golden Gate cloning, such that each sgRNA was controlled by a different Pol III promoter and fused to the optimized sgRNA constant region described in Chen et al. [91]. To this end, the sgRNA constant region of the lentiGuide-puro sgRNA expression plasmid (Addgene 52,963, [92]) was exchanged for the optimized version, thus generating the vector SP199 and the vector was digested with BsmBI (NEB) overnight at 37 °C and gel-purified. Two fragments were synthesized as gene blocks (IDT) containing the optimized sgRNA constant region (handle) coupled to the mU6 or hH1 promoter sequences. These fragments were then amplified with primers that contained part of the sgRNA sequence and a BsmBI restriction site (primer sequences can be found in Additional file 9: Table S8) and purified using the gel and PCR purification kit (Macherey&Nagel). The vector (100 ng) and two fragments were ligated in an equimolar ratio in a Golden Gate reaction with T4 ligase and the BsmbI isoschizomer Esp3I for 20 cycles (5 min 37 °C, 20 min 20 °C) and transformed into NEB Stable competent E. coli [93]. Successful assembly was verified by Sanger sequencing.

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