Plasmid MLM3636, expressing single-guide RNAs in mammalian cells, was a kind gift from Keith Joung (Addgene plasmid # 43860). Plasmid pMLM2.0 was constructed from MLM3636 by replacing the sgRNA expression cassette with the sgRNA2.0 expression cassette of the lentiviral plasmid sgRNA (MS2)_zeo backbone (Addgene plasmid # 61427) [26 (link)]. Stretches of 20 bp sgRNA to target UCHL1 were designed using the online tool (http://crispr.mit.edu/ ). Double-stranded oligonucleotides containing these targeting sites (listed in Supplemental Table 2) were cloned into BsmBI-digested pMLM2.0.
The plasmid containing the gene of the mammalian codon-optimized dCas9-VP64 activator (a tetramer of the viral VP16 transcriptional activator) was a kind gift from Keith Joung (Addgene; pMLM3705, #47754). An additional multiple-cloning site was inserted by replacing the VP64 coding sequence in dCas9-VP64 with a sequence containing a PacI restriction site, the new plasmid was referred to as No Effector Domain, pdCas9-NED (Addgene #109358) [53 (link)].
Plasmids expressing C-terminally mCherry-tagged dCas9-MSssI (Q147L/E186A) in mammalian cells were constructed as follows [54 (link)]: to create in-frame fusion between dCas9-MSssI and the P2A-mCherry-tag, the double-stranded oligonucleotide AK473-AK474 (5’-CGCGCCCAT ATGTTAATTAACAATTAA/5’-CCGGTTAATTGTTAATTAACATATGGG) was inserted between the SgsI (AscI) and BshTI (AgeI) restriction sites of pSYC-187 (Addgene#74,794). Insertion of AK473-AK474 preserved the flanking restriction sites, introduced a unique PacI site (underlined) and an in-frame stop codon. To abolish the stop codon, a short oligo-duplex (AK481-AK482, 5’-TAAGGTACCGA/5’-CCGGTCGGTACCTTAAT) was cloned between the PacI and the BshTI (AgeI) sites to obtain the plasmid pMCS-P2A-mCherry. (MCS stands for a sequence containing several restriction sites.) The SgsI (AscI) and Eco105I (SnaBI) fragment encoding the ‘MCS-P2A-mChery’ fragment was excised from pMCS-P2A-mCherry and cloned between the SgsI (AscI) and MssI (PmeI) sites of pdCas9-NED. The resulting plasmid pM-dCas9-(NED)-P2A_mCherry encodes a dCas9-P2A-mCherry fusion. The coding sequences of the MSssI (Q147L) and MSssI (E186A) variants were inserted, on SgsI-PacI fragments, between the SgsI and PacI sites of pdCas9-(NED)-P2A_mCherry. The new plasmids named pM-dCas9-MsssI (Q147L)-P2A-mCherry and pM-dCas9-MsssI (E186A)-P2A-mCherry express the respective dCas9-MSssI variant carrying the self-cleavable mCherry-tag. The catalytic domain of H3K27 histone methyltransferase enhancer of zeste homolog 2 (EZH2) was amplified with overhangs containing AscI and PacI restriction sites by PCR from pdCas9-EZH2. The EZH2 catalytic domain was subcloned into the pM-dCas9-NED-P2A-mCherry plasmid to yield pM-dCas9-EZH2-P2A-mCherry. The P2A-mCherry coding sequence was from the plasmid pSYC-187 [55 (link)], which was a kind gift from Seok-Yong Choi (Addgene plasmid # 74794). The catalytic domain of PRDM9 and DOT1L from dCas9-PRDM9 and dCas9-DOT1L, constructed as previously described [47 (link)], were subcloned into the pM-dCas9-NED-P2A-mCherry plasmid.
UCHL1 full-length cDNA (669 bp) was amplified from BEAS-2B cells and inserted into pcDNA4/HisMaxA between the BamHI and XbaI sites to generate the pcDNA4-UCHL1. Structure of the recombinant plasmids was confirmed by sequencing.
The plasmid containing the gene of the mammalian codon-optimized dCas9-VP64 activator (a tetramer of the viral VP16 transcriptional activator) was a kind gift from Keith Joung (Addgene; pMLM3705, #47754). An additional multiple-cloning site was inserted by replacing the VP64 coding sequence in dCas9-VP64 with a sequence containing a PacI restriction site, the new plasmid was referred to as No Effector Domain, pdCas9-NED (Addgene #109358) [53 (link)].
Plasmids expressing C-terminally mCherry-tagged dCas9-MSssI (Q147L/E186A) in mammalian cells were constructed as follows [54 (link)]: to create in-frame fusion between dCas9-MSssI and the P2A-mCherry-tag, the double-stranded oligonucleotide AK473-AK474 (5’-CGCGCCCAT ATGTTAATTAACAATTAA/5’-CCGGTTAATTGTTAATTAACATATGGG) was inserted between the SgsI (AscI) and BshTI (AgeI) restriction sites of pSYC-187 (Addgene#74,794). Insertion of AK473-AK474 preserved the flanking restriction sites, introduced a unique PacI site (underlined) and an in-frame stop codon. To abolish the stop codon, a short oligo-duplex (AK481-AK482, 5’-TAAGGTACCGA/5’-CCGGTCGGTACCTTAAT) was cloned between the PacI and the BshTI (AgeI) sites to obtain the plasmid pMCS-P2A-mCherry. (MCS stands for a sequence containing several restriction sites.) The SgsI (AscI) and Eco105I (SnaBI) fragment encoding the ‘MCS-P2A-mChery’ fragment was excised from pMCS-P2A-mCherry and cloned between the SgsI (AscI) and MssI (PmeI) sites of pdCas9-NED. The resulting plasmid pM-dCas9-(NED)-P2A_mCherry encodes a dCas9-P2A-mCherry fusion. The coding sequences of the MSssI (Q147L) and MSssI (E186A) variants were inserted, on SgsI-PacI fragments, between the SgsI and PacI sites of pdCas9-(NED)-P2A_mCherry. The new plasmids named pM-dCas9-MsssI (Q147L)-P2A-mCherry and pM-dCas9-MsssI (E186A)-P2A-mCherry express the respective dCas9-MSssI variant carrying the self-cleavable mCherry-tag. The catalytic domain of H3K27 histone methyltransferase enhancer of zeste homolog 2 (EZH2) was amplified with overhangs containing AscI and PacI restriction sites by PCR from pdCas9-EZH2. The EZH2 catalytic domain was subcloned into the pM-dCas9-NED-P2A-mCherry plasmid to yield pM-dCas9-EZH2-P2A-mCherry. The P2A-mCherry coding sequence was from the plasmid pSYC-187 [55 (link)], which was a kind gift from Seok-Yong Choi (Addgene plasmid # 74794). The catalytic domain of PRDM9 and DOT1L from dCas9-PRDM9 and dCas9-DOT1L, constructed as previously described [47 (link)], were subcloned into the pM-dCas9-NED-P2A-mCherry plasmid.
UCHL1 full-length cDNA (669 bp) was amplified from BEAS-2B cells and inserted into pcDNA4/HisMaxA between the BamHI and XbaI sites to generate the pcDNA4-UCHL1. Structure of the recombinant plasmids was confirmed by sequencing.
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