DpnI (10 U/µL)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	We added 0.8 μl 10 x FD buffer and 0.2 μl DpnI (Thermo Scientific) to the final PCR product, and the mix was directly transformed into E. coli strain DH5α. Three single colonies were cultured, and plasmids extracted using the GeneJET Plasmid Miniprep Kit (Thermo Scientific), followed by Sanger-sequencing to confirm sgRNA integration. sgRNA sequences and vectors are listed in Additional file 5: Table S3.

Protocol tips

We added 0.8 μl 10 x FD buffer and 0.2 μl DpnI (Thermo Scientific) to the final PCR product, and the mix was directly transformed into E. coli strain DH5α. Three single colonies were cultured, and plasmids extracted using the GeneJET Plasmid Miniprep Kit (Thermo Scientific), followed by Sanger-sequencing to confirm sgRNA integration. sgRNA sequences and vectors are listed in Additional file 5: Table S3.

Publication protocol

Protospacer target sequences were designed with CRISPR-P [32]. In most cases, the suggestions with the highest scores were used. In cases where there was only a small number of automatic suggestions, sequences were chosen manually. Target sequences were inserted into the sgRNA shuffle-in vectors using overlapping PCR with primers that contained 20-bp vector sequences plus 20-bp guide RNA sequences. For example, forward primer with 5′ NV1NNNNV17NV18NV19NV20 + 20 bp-sgRNA, and reverse primer with 20 bp-sgRNA + NV21NV22NV23NV24NNNNV40 5′ were used for inserting a 20 bp-sgRNA between positions NV20 and NV21 of vector sequence NV1NNNNNNNV20NV21NNNNNNV40. The overlapping PCR reaction (10 μl) was set up as follows (final concentrations in brackets): Q5 Hi-Fidelity polymerase reaction buffer (1x, Thermo Scientific), dNTPs (200 μM), forward and reverse primers (0.2 μM each), ~60 ng vector as template, 0.1 μl Q5 polymerase, and ddH2O to 10 μl. PCR was according to the manual for Q5 polymerase (Thermo Scientific). We added 0.8 μl 10 x FD buffer and 0.2 μl DpnI (Thermo Scientific) to the final product, and the mix was directly transformed into E. coli strain DH5α. Three single colonies were cultured, and plasmids extracted using the GeneJET Plasmid Miniprep Kit (Thermo Scientific), followed by Sanger-sequencing to confirm sgRNA integration. sgRNA sequences and vectors are listed in Additional file 5: Table S3.

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