EagI-HF®

Protocol tips

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	The thereby assembled complementation cassette was gel-purified, verified by sequencing and further cleaved by EagI-HF (NEB) and SalI-HF (NEB) following manual’s instructions. Analogously, respective pENTRY-miniCR-constructs (see above) were cleaved by the same enzymes, respectively, as a EagI and a SalI restriction site are sequentially located 5 bp upstream of the truncated leader promoter (cf. Fig. 2a).

Protocol tips

The thereby assembled complementation cassette was gel-purified, verified by sequencing and further cleaved by EagI-HF (NEB) and SalI-HF (NEB) following manual’s instructions. Analogously, respective pENTRY-miniCR-constructs (see above) were cleaved by the same enzymes, respectively, as a EagI and a SalI restriction site are sequentially located 5 bp upstream of the truncated leader promoter (cf. Fig. 2a).

Publication protocol

For complementation constructs, the slaB gene of S. islandicus M.16.4 (M.16.4_1762, DNA, and S. solfataricus were aligned, which revealed M.16.4 slaB 4 and 10 mismatches in complementary regions of protospacers PS2 and PS3, respectively (cf. Fig. 2b). As mismatches were scattered throughout PS3, and PS2 exhibited two mismatches towards the 3′-end, which is considered important for target cleavage50, slaB-M.16.4 was considered divergent enough to avoid cross-targeting of simultaneously expressed SB2 and SB3-crRNAs. For creation of complementation cassettes, first the araS promoter of S. solfataricus P234 was PCR-amplified (Phusion Polymerase, ThermoFisher, Scientific) using araFW_EagI and araOH_MSlaB_RV primers (Supplementary Table 1), which added an EagI recognition site to the 5′ and an overhang consisting of the first 25 nt of the slaB-M.16.4 sequence (see below) to the 3′-end of the araS sequence, respectively. The sequence chosen for slaB-M.16.4 expression contained the slaB-M.16.4 coding sequence (CDS), as well as the 11 bp 5′-UTR containing a RBS38, as well as the putative terminator sequence downstream to the CDS (genome coordinates: 1615528–1614238, GenBank: CP001402.1). This region was amplified (DNA extracted from S. islandicus M.16.4 obtained from the Rachel Whitaker Laboratory) using primers SlaBM164_FW and SlaBM164_SalI_RV, thereby adding a SalI restriction site to its 3′-end (Supplementary Table 1). AraS and slaB-M.16.4 PCR products were gel-purified (Monarch Gel purification kit, NEB) and subsequently fused at the homologous overlap region using primers araFW_EagI and SlaBM164_SalI_RV, in a touch-down PCR reaction (Supplementary Table 1). The thereby assembled complementation cassette was gel-purified, verified by sequencing and further cleaved by EagI-HF (NEB) and SalI-HF (NEB) following manual’s instructions. Analogously, respective pENTRY-miniCR-constructs (see above) were cleaved by the same enzymes, respectively, as a EagI and a SalI restriction site are sequentially located 5 bp upstream of the truncated leader promoter (cf. Fig. 2a). Finally, the complementation cassette was ligated upstream to the miniCR cassette into the respective pENTRY vectors (Quick-Ligase, NEB), generating ENTRY-SB2-CC, pENTRY-SB3×6-CC, and pENTRY-Ctrl-CC, respectively which were recovered by E. coli propagation (One Shot TOP 10, ThermoFisher Scientific).

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