Fncas12a

LbCas12a (Cpf1), AsCas12a, FnCas12a and RNA inhibitor were purchased from New England Biolabs Co. Ltd (NEB, USA). N,N,N',N'-Tetramethylethylenediamine (TEMED), urea, ammonium persulphate (APS), 40% acrylamide/bis-acrylamide (19:1), Tris borate EDTA (TBE), 2 × TBE–urea sample buffer and Ezup cfDNA Extraction Kit were obtained from Sangon Biotech Co. Ltd (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM), Minimum Essential Medium (MEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Gibco, USA). Trypsin and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, USA). Human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa) and human embryonic kidney cancer cell line (HEK293T) were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). To create and maintain a ribonuclease-free environment, all the solutions containing RNAs were prepared with RNase-free water.

In-vitro digestion reactions were carried out with three different types of the Cas12a family (LbCas12a, AsCas12a, and FnCas12a were purchased from New England Biolabs Inc. or purified in the lab, Integrated DNA Technologies Inc., and abm®, respectively) and a wide array of modified crRNAs (purchased from DNA Technologies Inc.). Cas12a and crRNA were mixed with a 1:1 ratio (100 nM:100 nM) in 1× NEBuffer 2.1 and pre-incubated at 25 °C for 15 min to promote the ribonucleoprotein complex formation. DNA activator (final concentration of 7 nM) was then added to the mixture to produce a total volume of 30 μl and incubated at 37 °C for 30 min^{19 (link)}. The sample was then analyzed in either 1% agarose gel (for GFP fragments amplified from the pEGFP-C1 plasmid) pre-stained with either SYBER Gold (Invitrogen), GelRed (Biotium Inc.), or premade 15% Novex^TM TBE-Urea Gel (Invitrogen).