• A novel cloning method eliminates the need for PCR and annealing steps, using a single oligo for short DNA fragment insertion.
• The method leverages host cell DNA polymerase for complementary strand synthesis, with T4 DNA polymerase supplementation enhancing efficiency.
• Cloning time is reduced to under 1 hour, significantly faster than traditional methods requiring 2+ hours.
• The technique is broadly applicable for cloning shRNA and sgRNA, streamlining gene knockdown and CRISPR/Cas9 genome editing workflows.