AgeI-HF®

Protocol tips

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	The backbone was digested with AgeI-HF (NEB, R3552S). Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. For cloning of single gRNAs into the lentiviral backbone, gRNA sequences were ordered as strings (see Supplementary Table 3). Strings were amplified using the libgen_fwd and libgen_rev primers. The PCR mix contained 0.1 ng DNA template, 25 µl 2x Phusion High-Fidelity PCR Master Mix with HF Buffer, 0.5 µl of each primer (100 µM), in a total reaction volume of 50 µl. The backbone was digested with AgeI-HF. Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. Transformation, PCR and plasmid preparations were done according to routine laboratory practice. gRNA sequences are listed in Supplementary Table 3.

Protocol tips

The backbone was digested with AgeI-HF (NEB, R3552S). Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. For cloning of single gRNAs into the lentiviral backbone, gRNA sequences were ordered as strings (see Supplementary Table 3). Strings were amplified using the libgen_fwd and libgen_rev primers. The PCR mix contained 0.1 ng DNA template, 25 µl 2x Phusion High-Fidelity PCR Master Mix with HF Buffer, 0.5 µl of each primer (100 µM), in a total reaction volume of 50 µl. The backbone was digested with AgeI-HF. Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. Transformation, PCR and plasmid preparations were done according to routine laboratory practice. gRNA sequences are listed in Supplementary Table 3.

Publication protocol

gRNA targeting sequences were designed using a free online platform (www.benchling.com) that employs a published algorithm for gRNA binding efficiency51. Specificity scores above 40 were set as requirements, and only sequences with a 5’G were considered in order to fit the requirements of the human U6 promoter. gRNA sequences were designed in the region starting 250 bp upstream of the transcription start site (TSS) up to the TSS. For the STAgR constructs, the gRNAs of each pair were required to be at least 100 bp apart to avoid spatial hindrance. STAgR plasmids were generated using the protocol provided by Breunig et al., 201824. For lentiviral gRNA plasmids, the vector pLKO.1 (Addgene plasmid 10878) was modified as described in Koeferle et al.52. For subcloning of STAgR constructs into the modified pLKO1 backbone, the gRNA cassettes were amplified by PCR using the amp_gRNA_pLKO_fwd and amp_gRNA_rev primers. 25 μl Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531S), 0.5 μl of each primer (100 μM), 1 ng template in a total reaction volume of 50 μl. The backbone was digested with AgeI-HF (NEB, R3552S). Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. For cloning of single gRNAs into the lentiviral backbone, gRNA sequences were ordered as strings (see Supplementary Table 3). Strings were amplified using the libgen_fwd and libgen_rev primers. The PCR mix contained 0.1 ng DNA template, 25 µl 2x Phusion High-Fidelity PCR Master Mix with HF Buffer, 0.5 µl of each primer (100 µM), in a total reaction volume of 50 µl. The backbone was digested with AgeI-HF. Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. Transformation, PCR and plasmid preparations were done according to routine laboratory practice. gRNA sequences are listed in Supplementary Table 3.

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