4. Vortex.
5. PCR tubes (eight-strip format).
6. Multichannel pipette (eight channels).
7. Thermal cycler.
8. Tabletop centrifuge for PCR tubes (eight-strip format or
96-well plate).
9. Magnetic separator for PCR tubes (e.g., MagWell™ Magnetic
Separator 96 from EdgeBio, see Note 3).
10. Razor blade.
11. Nylon filter of 50 μm.
12. Flow cytometer (e.g., CyFlow Space from Sysmex).
13. Ultrafine point forceps (Dumont, see Note 4).
2.5 Reagents
All solutions are prepared with Milli-Q water.
2.5.1 MultiSite-Puchta
Vector Construction for One
or Two gRNAs
1. BpiI (10 U/μL; e.g., from Thermo Scientific, see Note 5).
2. T4 DNA ligase (5 U/μL).
3. 10Â T4 DNA ligase buffer (containing 10 mM ATP, see Note 6).
4. 1 mg/mL bovine serum albumin (BSA).
5. LR Clonase II.
6. LR Clonase II Plus.
7. Proteinase K (2 μg/μL).
8. 100 mg/mL carbenicillin, sterile-filtered.
9. 50 mg/mL kanamycin, sterile-filtered.
10. 50 mg/mL spectinomycin, sterile-filtered.
11. LB medium.
12. Plasmid miniprep kit.
13. Oligonucleotides for colony-PCR and Sanger sequencing can
be found in Table 4.
2.5.2 Modular Vector
Construction for Up to 12
gRNAs
1. BbsI-HF (20,000 U/mL (e.g., from New England Biolabs, see
Note 5).
2. BsaI (20,000 U/mL).
3. NheI (20,000 U/mL).
4. T4 DNA ligase (400,000 U/mL, see Note 6).
5. 10Â Recommended restriction enzyme buffer (containing
1 mg/mL BSA).
6. 10 mM ATP.
7. 100 mg/mL carbenicillin, sterile-filtered.
8. 50 mg/mL spectinomycin, sterile-filtered.
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