Ascas12a

Firstly, a preassembly step was conducted by incubating AsCas12a (IDT, Inc, #10001272) at a concentration of 50 nM with 62.5 nM crRNA at room temperature for 10 min. Next, the AsCas12a-crRNA complexes were combined with 1 μL of binding buffer (New England BioLabs, #B6002S), 4.5 μL of biotinylated quencher probes, and 3.625 μL of nuclease-free water. Following this, 2 μL of target DNA was added, initiating the CRISPR reaction, and allowed to incubate at 37 °C for a duration of 2 h.^{6 (link),51 (link)}

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).