1. T75 cell culture flasks (75 cm
2 ) (Corning, cat. no. 431464 U).
2. McCoy’s 5a or DMEM medium supplemented with 10% fetal
bovine serum (FBS), 4 mM GlutaMAX™ (Gibco, cat.
no. 35050061) and 1% PenStrep (Gibco, cat. no. 15140-122).
3. Phosphate-buffered saline (PBS).
4. Trypsin–EDTA solution (0.025% w/v Trypsin, 0.05 mM
EDTA in 1Â PBS) (Gibco, cat. no. R001100).
2.1 Reagents
for the Preparation
of sgRNA Expression
Plasmids
1. Oligonucleotides for preparing (sgRNA-FW, sgRNA-RV) and
sequencing (U6-seq) the single guide RNA expression plasmids (sgRNA) (Table 1).
2. Guide RNA expression plasmid: MLM3636 (Addgene plasmid
#43860).
3. T4 DNA ligase with buffer for the preparation of the guide
RNA expression plasmid (New England BioLabs, cat.
no. M0202S).
4. BsmBI restriction enzyme (New England BioLabs, cat.
no. R0580S).
Table 1
XPB locus
U-2 OS
XPB::GFP
Chromosome/gene
Chr. 2/XPB
Homology arms
5
0 Chr.2: 127257599–127257872 (hg 38)
3
0 Chr.2: 127257596–127256813 (hg 38)
sgRNA
5
0 TAGGAAATGATGCTTAGGCAggg 3
0
Selection marker
Puromycin
sgRNA-FW
5
0 ACACCG TAGGAAATGATGCTTAGGC G 3
0
sgRNA-RV
5
0 AAAAC TGCCTAAGCATCATTTCCTA CG
U6-seq
5
0 CAGGGTTATTGTCTCATGAGCGG-3
0
XPB-FW
5
0 AGACAGTAAGCGATCTGTAAACA 3
0
XPB-RV
5
0 ACCCCACTCCCCAAAAAGTT 3
0
XPB-FW2
5
0 TCCTCTTCTTTCAGGTGTGGA 3
0
GFP-RV
5
0 GAACTTCAGGGTCAGCTTGC 3
0
Puro-FW
5
0 GCAACCTCC CCTTCTACGAG 3
0
XPB-RV2
5
0 GCGAATATGCCTTATGTGTG 3
0
Target sequence for the sgRNA used for editing the C-terminus of the XPB gene. Online web applications such as
CRISPOR (http://crispor.tefor.net/) aimed at optimizing CRISPR knock-in tag experimentation identify and rank
potential guide RNAs in an input sequence. Oligonucleotides for sense and antisense strands designed with CRISPOR
software for cloning into MLM3636 (sgRNA-FW and sgRNA-RV) as well as for genomic screening (XPB-FW, RV) are
detailed. Efficient transcription from the U6 promoter requires a 5
0 G (underlined)
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
41
2 ) (Corning, cat. no. 431464 U).
2. McCoy’s 5a or DMEM medium supplemented with 10% fetal
bovine serum (FBS), 4 mM GlutaMAX™ (Gibco, cat.
no. 35050061) and 1% PenStrep (Gibco, cat. no. 15140-122).
3. Phosphate-buffered saline (PBS).
4. Trypsin–EDTA solution (0.025% w/v Trypsin, 0.05 mM
EDTA in 1Â PBS) (Gibco, cat. no. R001100).
2.1 Reagents
for the Preparation
of sgRNA Expression
Plasmids
1. Oligonucleotides for preparing (sgRNA-FW, sgRNA-RV) and
sequencing (U6-seq) the single guide RNA expression plasmids (sgRNA) (Table 1).
2. Guide RNA expression plasmid: MLM3636 (Addgene plasmid
#43860).
3. T4 DNA ligase with buffer for the preparation of the guide
RNA expression plasmid (New England BioLabs, cat.
no. M0202S).
4. BsmBI restriction enzyme (New England BioLabs, cat.
no. R0580S).
Table 1
XPB locus
U-2 OS
XPB::GFP
Chromosome/gene
Chr. 2/XPB
Homology arms
5
0 Chr.2: 127257599–127257872 (hg 38)
3
0 Chr.2: 127257596–127256813 (hg 38)
sgRNA
5
0 TAGGAAATGATGCTTAGGCAggg 3
0
Selection marker
Puromycin
sgRNA-FW
5
0 ACACCG TAGGAAATGATGCTTAGGC G 3
0
sgRNA-RV
5
0 AAAAC TGCCTAAGCATCATTTCCTA CG
U6-seq
5
0 CAGGGTTATTGTCTCATGAGCGG-3
0
XPB-FW
5
0 AGACAGTAAGCGATCTGTAAACA 3
0
XPB-RV
5
0 ACCCCACTCCCCAAAAAGTT 3
0
XPB-FW2
5
0 TCCTCTTCTTTCAGGTGTGGA 3
0
GFP-RV
5
0 GAACTTCAGGGTCAGCTTGC 3
0
Puro-FW
5
0 GCAACCTCC CCTTCTACGAG 3
0
XPB-RV2
5
0 GCGAATATGCCTTATGTGTG 3
0
Target sequence for the sgRNA used for editing the C-terminus of the XPB gene. Online web applications such as
CRISPOR (http://crispor.tefor.net/) aimed at optimizing CRISPR knock-in tag experimentation identify and rank
potential guide RNAs in an input sequence. Oligonucleotides for sense and antisense strands designed with CRISPOR
software for cloning into MLM3636 (sgRNA-FW and sgRNA-RV) as well as for genomic screening (XPB-FW, RV) are
detailed. Efficient transcription from the U6 promoter requires a 5
0 G (underlined)
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
41