Skip to main content
. 2022 Feb 8;10(2):e003804. doi: 10.1136/jitc-2021-003804

Figure 5.

Figure 5

Gene knock-in (KI) targeted to the CD38 locus results in highly efficient concurrent gene disruption and insertion. (A) Gene KI strategy to insert a truncated CD34 cassette into the CD38 locus using Cas9/RNP and rAAV6. One of the CD34 cassettes contained an internal EF1α promoter and the other had a self-cleaving P2A peptide sequence in place of a promoter. (B) Representative gel image of PCR to detect site-specific integration of the CD34 transgene within the CD38 locus. (C) Representative flow cytometry plots of CD38 and CD34 expression 7 days after gene KI targeted to the CD38 locus. (D) CD34 expression assessed by percent positive and percent positive NK cells over time following Cas9/RNP electroporation and/or rAAV6 infection examined by flow cytometry (n=5 donors for CD38KO/CD34KI groups and n=3 donors for all other groups). (E) Efficiency of 2-in-1 KO/KI determined by CD38 and CD34 expression in control and edited NK cells, examined by flow cytometry (n=3 donors). Statistics determined with the Student’s t-test, two tailed, **p<0.01, ****p<0.0001. KO, knockout; WT, wild type; NK, natural killer; LHA, left homology arm; RHA, right homology arm.