Individual perfectly matched or mismatched sgRNAs were cloned essentially as described previously18 (link). Briefly, two complementary oligonucleotides (Integrated DNA Technologies), containing the targeting region as well as overhangs matching those left by restriction digest of the backbone with BstXI and BlpI, were annealed and ligated into an sgRNA expression vector digested with BstXI (NEB or Thermo Fisher Scientific) and BlpI (NEB) or Bpu1102I (Thermo Fisher Scientific). The ligation product was transformed into Stellar chemically competent E. coli cells (Takara Bio) and plasmid was prepared following standard protocols.
Protocol detail
Cloning of sgRNA Expression Vectors
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Corresponding Organization :
Other organizations : University of California, San Francisco, Howard Hughes Medical Institute
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Variable analysis
independent variables
- Individual perfectly matched or mismatched sgRNAs
- Not explicitly mentioned
- Restriction enzymes used for cloning (BstXI and BlpI or Bpu1102I)
- Competent E. coli cells used for transformation (Stellar chemically competent cells)
- Positive control: Not mentioned
- Negative control: Not mentioned
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