Construction of sgRNA expression cassette.

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Construction of sgRNA expression cassette. Construction of sgRNA expression cassette. Three DNA synthesis steps fuse DNA fragments comprising the SNR52 promoter and the sgRNA scaffold. Chimeric primers 2 and 3 carry 20 complementary bases of guide sequence. In the first step, PCR is used to create two segments of the sgRNA gene. The SNR52 promoter is amplified with primers 1 and 2; the sgRNA scaffold is amplified with primers 3 and 4. In the second step, primer extension is used to fuse the two PCR products, with chimeric extensions acting as primers. In the third step, PCR with nested primers 5 and 6 is used to amplify the final sgRNA expression cassette. Kyunghun Min et al. mSphere 2016; doi:10.1128/mSphere.00130-16