Somatic hypermutation of J3-CH2 HCAb in immunized human tonsil organoids
Human tonsil B cells were engineered with an anti-HIV J3-CH2 HCAb using spCas9 RNPs and AAV6 homology donors. Engineered cells were then mixed with total tonsil mononuclear cells to establish a tonsil organoid containing reprogrammed human B cells. Organoids were then immunized with recombinant HIV gp120 with AdjuPhos adjuvant. Twelve days later, bulk DNA was harvested from organoids and a nested PCR approach was used to amplify integrated J3 sequences, tag with 3' UMIs, and then add 5' barcodes and prepare amplicons for 2x250 paired end Illumina sequencing to investigate somatic hypermutation (SHM) of the engineered J3 cassette.