Lambda phage

One hundred microliters of overnight host bacterial culture were mixed with 50 µL of phage stock solution, and then 4 mL of prewarmed semisolid overlay agar (LB broth with 0.6% agar) was added to spread the mixture on LB agar plates. After overnight incubation at 37 °C, 3–5 mL of SM preservative fluid (2 g/L MgSO₄·7H₂O, 5.8 g/L NaCl, 50 mL 1 M Tris-HCl, pH 7.4) was added to the plates. Then, the supernatant solution was collected and filtered through a 0.22 µm filter, and the fresh phage stock solutions were stored at 4 °C. The lambda phage (purchased from New England Biolabs) used in this study carries a mutation in the cI gene encoding a phage repressor, which ensures a default lytic pathway at the temperatures of 37 °C and above, and switches to a lysogenic pathway when temperature drops to e.g. 30 °C^{71 (link)} (Supplementary Table 2). In our experiments, the cultivation temperature of both host strains and lambda phage was at 37 °C, and thus phage lambda undergoes the lytic pathway. As shown by Gordeeva et al.^{72 (link)}, lambda cI₈₅₇ phage is capable of infecting E. coli cells even in the absence of induction.

First, 400 ng of Cas9nickase—Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1 × NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37 °C for 15 min to form Cas9-Ribonucleoprotein complex. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37 °C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100–300 µM dNTPs, and 1 × Thermopol (NEB) at 72 °C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation.