5. LB and LB agar medium, ampicillin, competent DH5α E. coli
cells (New England BioLabs, cat. no. C2987I).
6. Plasmid DNA Mini and Midiprep purification kits (MachereyNagel™ NucleoSpin Plasmid QuickPure™ Kit and MachereyNagel™ NucleoBond™ Xtra Midi).
2.2 Reagents
for Nucleofection
and Assessment
of Gene Editing
2.2.1 Nucleofection
with AMAXA Nucleofector
Machine
1. Expression plasmid for SpCas9: JDS246 (Addgene plasmid
#43861).
2. Expression plasmid for sgRNA (Addgene plasmid #43860)
(from Subheading 3.1, step 4).
3. AMAXA nucleofector machine
® (Lonza) and consumables
including cuvettes (Lonza, cat. no. AAB-1001), pipettes, and
solution V kit (Lonza, cat. no. VVCA-1003).
4. Tissue
culture
plate,
6
wells
(Eppendorf,
cat.
no. EP0030720121).
2.2.2 Pool and Clone
Analysis
1. QuickExtract DNA solution (Viagen, cat. no. 302C).
2. Oligonucleotides for PCR amplification and sequencing (see
Table 1).
3. Phusion high-fidelity DNA polymerase with buffer and dNTPs
(New England BioLabs, cat. no. M053OS).
4. PCR isolation kit (Macherey-Nagel™ NucleoSpin™ Gel and
PCR Clean-up Kit).
2.3 Reagents
for Gene Editing
and FACS Sorting
2.3.1 Nucleofection
and Antibiotic Selection
In addition to the reagents described in Subheading 2.2.1
1. Donor plasmid (from Subheading 3.3, step 2).
2. Puromycin dihydrochloride (InvivoGen, cat. no. ant-pr-1).
2.3.2 FACS Sorting
Sorting of GFP-fluorescent cells was carried out on a Melody FACS
(BD), and analysis of GFP-positive cells was carried on an Accuri
C6 analyzer (BD). You will need:
1. Cytometry Falcon tubes (Becton Dickinson).
2. Tissue culture plate, 96 wells (Eppendorf, cat.
no. EP0030730119).
2.4 Reagents
for the Validation
of Selected Clones
2.4.1 Genotyping
of Single-Cell Clones
In addition to reagents described in Subheading 2.2.2, you will
need:
1. Tissue culture plates, 6 and 96 wells (Eppendorf, cat.
no. EP0030720121, EP0030730119).
2. Oligonucleotides from the inserted sequence and from the
target chromosomal locus.
42
Sylvain Geny et al.
cells (New England BioLabs, cat. no. C2987I).
6. Plasmid DNA Mini and Midiprep purification kits (MachereyNagel™ NucleoSpin Plasmid QuickPure™ Kit and MachereyNagel™ NucleoBond™ Xtra Midi).
2.2 Reagents
for Nucleofection
and Assessment
of Gene Editing
2.2.1 Nucleofection
with AMAXA Nucleofector
Machine
1. Expression plasmid for SpCas9: JDS246 (Addgene plasmid
#43861).
2. Expression plasmid for sgRNA (Addgene plasmid #43860)
(from Subheading 3.1, step 4).
3. AMAXA nucleofector machine
® (Lonza) and consumables
including cuvettes (Lonza, cat. no. AAB-1001), pipettes, and
solution V kit (Lonza, cat. no. VVCA-1003).
4. Tissue
culture
plate,
6
wells
(Eppendorf,
cat.
no. EP0030720121).
2.2.2 Pool and Clone
Analysis
1. QuickExtract DNA solution (Viagen, cat. no. 302C).
2. Oligonucleotides for PCR amplification and sequencing (see
Table 1).
3. Phusion high-fidelity DNA polymerase with buffer and dNTPs
(New England BioLabs, cat. no. M053OS).
4. PCR isolation kit (Macherey-Nagel™ NucleoSpin™ Gel and
PCR Clean-up Kit).
2.3 Reagents
for Gene Editing
and FACS Sorting
2.3.1 Nucleofection
and Antibiotic Selection
In addition to the reagents described in Subheading 2.2.1
1. Donor plasmid (from Subheading 3.3, step 2).
2. Puromycin dihydrochloride (InvivoGen, cat. no. ant-pr-1).
2.3.2 FACS Sorting
Sorting of GFP-fluorescent cells was carried out on a Melody FACS
(BD), and analysis of GFP-positive cells was carried on an Accuri
C6 analyzer (BD). You will need:
1. Cytometry Falcon tubes (Becton Dickinson).
2. Tissue culture plate, 96 wells (Eppendorf, cat.
no. EP0030730119).
2.4 Reagents
for the Validation
of Selected Clones
2.4.1 Genotyping
of Single-Cell Clones
In addition to reagents described in Subheading 2.2.2, you will
need:
1. Tissue culture plates, 6 and 96 wells (Eppendorf, cat.
no. EP0030720121, EP0030730119).
2. Oligonucleotides from the inserted sequence and from the
target chromosomal locus.
42
Sylvain Geny et al.