Generation of gRNA by overlap PCR

FP RP T7 promoter gRNA (20 bp) overlap gRNA scaffold

GAAATTAATACGACTCACTATAGG NNNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGC CAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAATAGTTGAACTTTTTCACCGTGGCTCAGCCACGAAAA

PCR

GAAATTAATACGACTCACTATAGG NNNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGC CAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAATAGTTGAACTTTTTCACCGTGGCTCAGCCACGAAAA

DNA Template for gRNA transcription In vitro transcription gRNA