Rosetta 2 de3 plyss singles competent cells

All BrCas12b variants were expressed in Rosetta 2(DE3)pLysS Singles Competent Cells (purchased from Millipore Sigma). Cells were kept frozen at −80°C until use. Cells were cultured as per supplier’s protocol with slight modifications. For transformation, frozen cells were thawed and then incubated with plasmids for 5 min on ice, heat-shocked for 30 s at 42°C in a water bath, placed back on ice for 2 min, and then cultured at 37°C in SOC medium (New England Biolabs) at 250 rpm for 1 h before spreading on antibiotic-containing agar plates. After spreading, the plates were incubated at 37°C for 12–48 h and then the colonies were picked, expanded in 10 mL Luria Broth (Fisher Scientific) containing the appropriate antibiotics, and sequenced by Sanger sequencing. Please refer to protein expression and purification section for more details.

Cas12a orthologs were expressed in Rosetta 2(DE3)pLysS Singles Competent Cells (Millipore Sigma Catalog #70956) following the supplier’s protocol. To initiate transformation, the frozen cells were thawed from −80°C, incubated with plasmids for 5 min on ice, heat-shocked for 30 s at 42°C in a water bath, incubated on ice for 2 min, and then cultured at 37°C in SOC medium (New England Biolabs Catalog #B9020) at 250 rpm for 1 h before being spread on antibiotic-containing agar plates. Post spreading, the plates were incubated at 37°C for 12–48 h, and subsequently, the colonies were selected, expanded in 10 mL Luria Broth (Fisher Scientific Catalog #BP9723) containing the appropriate antibiotics, and subjected to Sanger sequencing. For additional details, please consult the protein expression and purification section.
HEK293T cells were sourced from ATCC (Cat# CRL-3216) and were cultured in D10 medium (Dulbecco’s Modified Eagle’s Medium [DMEM, Gibco] supplemented with 1% Penicillin/Streptomycin and 10% FBS) at 37°C with 5% CO2. Cells intended for downstream transfection and nucleofection procedures were maintained within passages 5 to 15. Any cells beyond the 15th passage were disposed of.