T4 dna ligase

S. mutans mutant strains were constructed by PCR ligation mutagenesis (45 (link)) using the primers listed in Table S2. Briefly, 5′- and 3′-flanking regions of the target gene were amplified using chromosomal DNA of S. mutans UA159 as PCR templates. The flanking regions were ligated with an antibiotic marker cassette derived from pJL105 using T4 DNA ligase (Enzynomics, Daejeon, South Korea). The resulting products were transformed into S. mutans and the target gene was replaced with an antibiotic cassette via homologous recombination of the flanking regions. All cas genes in CRISPR1-Cas and CRISPR2-Cas were replaced with the spectinomycin cassette (designated ΔCR1cas) or kanamycin cassette (designated ΔCR2cas), respectively. A double-deletion mutant was constructed using the same method (ΔCRDcas). For fusion of the CRISPR promoter, the upstream region of each CRISPR locus was amplified with custom primers (Table S2) and cloned into the pMZ-lacZ integration vector (46 (link)), which carries the lacZ gene lacking a promoter and ribosomal binding site. The resulting construct was transformed into S. mutans to establish a promoter-lacZ fusion in a single copy of the chromosome by double-crossover homologous recombination, with mtlA-phnA genes serving as the integration site.

Each type of plasmid used in the Sniper-screen contains replication origins and resistance markers that are compatible with each other. (Fig. 1b) The ccdB plasmid (p11-lacY-wtx1) was a kind gift from the Zhao lab^{9 (link)}. It was double-digested with SphI and XhoI enzymes (Enzynomics), which was ligated to oligos (Cosmogenetech) containing target sequences (Supplementary Table 1) with T4 DNA ligase (Enzynomics). The sgRNA vector was constructed (Supplementary Figure 19) with a temperature-sensitive Psc101 replication origin^{12 (link)} (from pgrg36, a kind gift from Nancy Craig), tetR (from the tn10 locus of ElectroTen-Blue Electroporation Competent Cells, Agilent), a Kanamycin resistance marker, the pltetO1 promoter and the sgRNA sequence containing two BsaI sites (synthesized at Bioneer). The components were PCR-amplified and Gibson assembled (NEBuilder HiFi DNA Assembly kit, NEB). The guide RNA sequences to EMX1 with various mismatches (Supplementary Table 1) were cloned into the vector after BsaI digestion. The Cas9 library plasmid (Supplementary Figure 19) was derived from human codon-optimized WT-Cas9 (p3s-Cas9HC; Addgene plasmid #43945)^{37 (link)}, dual CMV-pltetO1 (synthesized at Bioneer) and the p15a replication origin and chloramphenicol resistance marker (from the PBLC backbone, Bioneer). The components were Gibson assembled.