Modular CRISPR-based Transcriptional Regulation

LentiCRISPR (Addgene, catalog no. 49535) was mutated at amino acid positions D10A and H840A by mutagenesis polymerase chain reaction (PCR) to construct Flag-dCas9-P2A-puro. Mouse TET1 catalytic domain (TET1CD) or p300CD was amplified from complementary DNA (cDNA) and subcloned into a MIGR vector. TET1CD or p300CD and internal ribosome entry site green fluorescent protein (IRES-GFP) sequences were amplified with a Gly–Gly–Gly–Gly–Ser linker and recombined into Flag-dCas9-P2A-puro instead of P2A-puro. Flag-dCas9-TET1CD or p300CD and IRES-GFP were recombined into pMXs-GW vectors. Amino acid sequences of each construct are detailed in the supplementary material (Additional file 1). For gRNA expression, DsRed was recombined into LentiCRISPR instead of Cas9-P2A-puro. Next, U6-gRNA-EFS-DsRed was recombined into CSII vector. Each gRNA expression vector was generated by the annealing of the oligonucleotides, followed by ligation into BsmBI-digested gRNA expression vectors based on CSII vector for lentiviral infection. In some cases, U6-gRNA-EFS-DsRed was recombined into pMXs-GW vectors for retroviral infection. gRNA off-target predictions were performed by CCTop—CRISPR/Cas9 target online predictor [41 (link)]. Max. total mismatches, core length, and max. core mismatches were set to 4, 12, and 2, respectively. Predicted off-target loci were listed in Additional file 2: Table S1.

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Okada M., Kanamori M., Someya K., Nakatsukasa H, & Yoshimura A. (2017). Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells. Epigenetics & Chromatin, 10, 24.

Publication 2017

LentiCRISPR

Amino acid Amino acid sequences Catalytic domain Cdna Crispr Gly gly gly gly Green fluorescent protein Infection Internal ribosome entry site Ligation Mouse Mutagenesis Oligonucleotides Polymerase chain reaction Retroviral infection Vectors

Corresponding Organization : Keio University

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Variable analysis

independent variables

Mutagenesis PCR to construct Flag-dCas9-P2A-puro
Amplification and subcloning of mouse TET1 catalytic domain (TET1CD) or p300CD into MIGR vector
Recombination of TET1CD or p300CD and IRES-GFP sequences into Flag-dCas9-P2A-puro
Recombination of Flag-dCas9-TET1CD or p300CD and IRES-GFP into pMXs-GW vectors
Recombination of DsRed into lentiCRISPR instead of Cas9-P2A-puro
Recombination of U6-gRNA-EFS-DsRed into CSII vector
Generation of gRNA expression vectors by annealing oligonucleotides and ligation into BsmBI-digested gRNA expression vectors based on CSII vector
Recombination of U6-gRNA-EFS-DsRed into pMXs-GW vectors

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive controls: None mentioned
Negative controls: None mentioned

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