To construct TALEN, TAL repeats were assembled by the modified Golden Gate assembly method (Sakuma et al., 2013 (link)).
To construct sgRNA vectors for CRISPR-Cas9, two oligonucleotides corresponding to the 5’-side of exon 4 of the XBP1 gene (5'-taggCTGCGGACTCAGAAGACC-3' and 5'-aaacGGTCTTCTGAGTCCGCAG-3'), and two oligonucleotides corresponding to the 3’-side of exon 4 of the XBP1 gene (5'-taggTGCCTCCGCAGCAGGTGC-3' and 5'-aaacGCACCTGCTGCGGAGGCA-3') were annealed and ligated into BsaI-digested DR274 vector (Addgene). sgRNA vectors were linearized with DraI, purified by phenol chloroform extraction, and used as template to synthesize sgRNAs using T7 RNA polymerase.
TALEN and Cas9(D10A) expressing vectors were linearized with NotI, purified by phenol chloroform extraction, and used as template to synthesize capped mRNAs using the Message mMachine SP6Kit (Life Technologies, Gaithersburg, MD).
Synthesized RNAs were purified by RNeasy MinElute (Qiagen, Germany) and microinjected into one-cell stage embryos at the concentration of 50 ng/μl for both left and right TALEN, 100 ng/μl for Cas9(D10A), and 25 ng/μl for both sense and antisense strand sgRNA of Cas9. Injection was performed as described previously (Ishikawa et al., 2011 (link)).
To construct sgRNA vectors for CRISPR-Cas9, two oligonucleotides corresponding to the 5’-side of exon 4 of the XBP1 gene (5'-taggCTGCGGACTCAGAAGACC-3' and 5'-aaacGGTCTTCTGAGTCCGCAG-3'), and two oligonucleotides corresponding to the 3’-side of exon 4 of the XBP1 gene (5'-taggTGCCTCCGCAGCAGGTGC-3' and 5'-aaacGCACCTGCTGCGGAGGCA-3') were annealed and ligated into BsaI-digested DR274 vector (Addgene). sgRNA vectors were linearized with DraI, purified by phenol chloroform extraction, and used as template to synthesize sgRNAs using T7 RNA polymerase.
TALEN and Cas9(D10A) expressing vectors were linearized with NotI, purified by phenol chloroform extraction, and used as template to synthesize capped mRNAs using the Message mMachine SP6Kit (Life Technologies, Gaithersburg, MD).
Synthesized RNAs were purified by RNeasy MinElute (Qiagen, Germany) and microinjected into one-cell stage embryos at the concentration of 50 ng/μl for both left and right TALEN, 100 ng/μl for Cas9(D10A), and 25 ng/μl for both sense and antisense strand sgRNA of Cas9. Injection was performed as described previously (Ishikawa et al., 2011 (link)).
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