Fig. 4.

Workflow of Random-PE in cultured mammalian cells. a. Diagram showing the design and screen of functional PE3 system targeting interest genes. b. Construction of PCR pegRNA random library. A forward primer located upstream of U6 promoter and a reverse primer containing complementary sequences to the sgRNA scaffold, HA, aimed random sequences, PBS, and U6 termination signal are used to amplify the existing sgRNA or pegRNA. c. Delivery of pegRNA library together with PE2 and nick sgRNA plasmids into interest cells. d. Detection of the desired insertion of random sequences in the genome. Genomic DNAs are extracted from transfected cells and subjected to PCR amplification using primers flanking the target region. The presence of the desired insertion of random sequences is detected by Sanger sequencing and quantified by HTS