Fig. 1

Schematic of the sgRNA Library Synthesis Method. Each step of the process is represented by a black arrow. Restriction enzyme binding sites are shown in black. Magnetic beads are represented as orange circles. (Step 1) DNA from an arbitrary source is fragmented by a restriction enzyme such as HpaII (CCGG) that contains the canonical PAM (NGG) in its recognition sequence. (Step 2) An adapter (blue) encoding a modified sgRNA sequence is ligated to the DNA fragments. The adapter is unphosphorylated, contains an MmeI recognition sequence, has a two base-pair, single-stranded overhang compatible with the fragmented DNA, and includes a long single-stranded overhang capable of hybridizing to a single-stranded oligo. (Step 3) A single-stranded capture oligonucleotide attached to a magnetic bead at the 3′ end is used to immobilize the adapter to magnetic beads through hybridization and excess DNA is washed away before digestion with MmeI to capture the spacer sequence. (Step 4) An unphosphorylated adapter containing the T7 promoter sequence (green) and a 3′ overhang containing all possible dinucleotides is ligated to the cleaved spacer sequence. (Step 5) The nicked library is detached from the beads by a strand displacing polymerase (yellow) and nicks are simultaneously repaired by the same mechanism. The detached double stranded oligonucleotides constitute an sgRNA library containing spacers targeting the DNA source and can be transcribed in vitro or cloned into plasmids for downstream applications.