Phanta max super fidelity dna polymerase p505

All plasmids and guide RNAs used in this study can be found in S1 Table. AsCas12a human expression plasmid pY010 was purchased from the nonprofit plasmid repository Addgene (Addgene plasmids #69982). All AsCas12a variants were generated by standard site-directed mutagenesis. In brief, using AsCas12a plasmid pY010 as the template and a pair of primers that one primer carrying site mutation nucleotide to amplify 500 bp fragments by PCR (Phanta MAX Super-Fidelity DNA Polymerase P505, Vazyme). After confirming that the bands were correct by agarose gel electrophoresis, the PCR products were purified. Next, taking 1,000 ng PCR products as the circling mutation primers and 100 ng AsCas12a plasmid pY010 as template to carry out PCR again. PCR products were purified and use DpnI to digest the original template at 37°C for 1 h, inactivated at 80°C for 20 min, take 10 μl digested products for transformation. The next day, single colonies were sent for sequencing. Oligonucleotide duplexes corresponding to spacer sequences were PCR amplified and cloned into pU6-As-crRNA plasmids (Addgene plasmids #78956) for U6 promoter-driven U6 promoter-driven transcription of As crRNAs (ClonExpress II One Step Cloning Kit C112, Vazyme).