Dna fragment purification kit ver 4

Bacterial genomic DNA was extracted using a TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China). Small-scale plasmid DNA preparations were generated using a E.Z.N.A™ Plasmid Miniprep Kit (Omega Bio-Tek, Norcross, GA, USA). The DNA fragments were amplified in a C1000™ Thermal Cycler using the 2×Taq Plus Master Mix (Vazyme, Jiangsu Sheng, China) or I-5^TM 2 ×High Fidelity Master Mix (MCLAB, South San Francisco, CA, USA). Purification of DNA fragments from the PCR reaction and the restriction digests were performed using the DNA Fragment Purification Kit Ver.4.0 (TaKaRa, Shiga, Japan). In-fusion segments were recombined to linearized pK18mobsacB (EcoRI/BamHI) using a ClonExpress II One Step Cloning Kit (Vazyme, Jiangsu Sheng, China). Restriction enzymes were purchased from TaKaRa. Point mutations were performed according to manual of Fast mutagenesis system Kit (TransGen Biotech, Beijing, China).

Genomic sequences with 5′-GN₁₉NGG-3′, where NGG is PAM, was screened and selected as candidate targets^{7 (link)}. The linearized pLS-HCas9 was generated using restriction enzymes BamHI and HindIII, and then was purified using DNA Fragment Purification Kit Ver.4.0 (TaKaRa, China). Target sequences were introduced into sgRNA expression cassettes by overlapping PCR. First round of PCR (20 μL) used four primers: U-F and gR-R (0.2 μM each) and two target sequence-containing chimeric primers U6T#+ and U6T#− (0.1 μM each) and PrimeSTAR MAX DNA Polymerase (TaKaRa, China) for 30 cycles (98 °C, 10 s; 58 °C, 5 s; 72 °C, 20 s). Second PCRs were set up with 0.5 μL of the PCR products and combinations of the primer pair U-Fs-BamHI/gR-R-HindIII (0.2 μM each) for In-fusion, and run for 30 cycles (98 °C, 10 s; 58 °C, 5 s; 72 °C, 30 s) to obtain a complete sgRNA expression cassette. The sequences of primers used in this study are listed in Supplementary, Table S1. The sgRNA expression cassette was purified and cloned into the binary vectors.