Figure 1. LIC based assembly strategy.

A schematic view of the LIC-based sgRNA cloning system is depicted. In brief, a backbone plasmid containing constant portions of a hybrid sgRNA downstream the U6 promoter is first linearized using ApaI and SpeI restriction enzymes. Subsequently, a T4 DNA polymerase chewback reaction in the presence of dTTP sets free long 5′ overhangs required for LIC. After annealing of the backbone plasmid to one constant as well as to one target-specific oligonucleotide, a 19 bp stretch remains single-stranded. Upon transformation into E. coli, a perfectly repaired, amplification-competent circular plasmid is obtained, containing the desired hybrid sgRNA sequence.