FIG. 6.

Using ErCas12a for targeted integration in human cells. (A) Schematic showing the workflow for targeted integration at AAVS1 in HEK293T cells. (B) Schematic of AAVS1 showing designed homology for precise integration using ErCas12a and the U-pre-crRNA to liberate the cassette. Green block, 5′ homology; blue block, 3′ homology. The PAM for ErCas12a targeting in the genome and donor is underlined. (C) Schematic for flow analysis after transfection of GeneWeld reagents for targeting AAVS1. (D) Bar graph showing the ratio of cells with GFP expression as determined by flow cytometry. Graph represents the mean ± SD. p-Values calculated with one-tailed Student's t-test. (E) Junction fragment gel showing the expected 5′ integration amplicon. No amplicon is seen when transfecting donor alone. (F) DNA sequencing of lanes in (E) showing a precise integration using the programmed homology.