DSB structure impacts DNA recombination leading to class switching and chromosomal translocations in human B cells
Fig 1
Cas9-mediated switching in human B cells.
(A) Schematic depicting Cas9 variants and orientation of sgRNAs required to generate blunt, 5’, and 3’DSBs. One sgRNA and wild-type Cas9 produces a blunt DSB, while paired sgRNAs and Cas9 D10A or Cas9 N863A give rise to 5’ or 3’DSBs, respectively. (B) Schematic depicting sgRNA target regions in the immunoglobulin heavy chain (IGH) locus on human chromosome 14 in normal BJAB cells and the resulting chromosome after Cas9-mediated switching. The sgRNAs in this experiment target S’μ, a non-repetitive region between the rearranged VDJ gene segment and the switch μ (Sμ) region. Cleavage at the S’μ_1 sgRNA target site with wild-type Cas9 produces a blunt DSB, while cleavage with the Cas9 nickases and sgRNAs S’μ_1 and _2 produces a staggered DSB with a 38 nt overhang. A similar strategy is applied using sgRNAs targeting S’α to produce blunt DSBs or staggered DSBs with a 63 nt overhang. Schematic is not to scale. (C) Percentage of IgA+ BJAB cells from blunt, 5’, and 3’DSBs measured by flow cytometry, 5 days post-transfection with Cas9/sgRNA plasmids. “-”, mock transfection. (D) Total resection of S’μ and S’α at S’μ-S’α junctions from blunt, 5’, and 3’DSBs. Dotted line denotes the total overhang length in S’μ plus S’α (101 nt). (E) Microhomology usage at S’μ-S’α junctions from blunt, 5’, and 3’DSBs. Black line denotes mean microhomology usage. (F) Percentage of IgA+ Ramos cells from blunt, 5’, and 3’DSBs measured by flow cytometry, 3 days post-transfection with Cas9/sgRNA plasmids. “-”, mock transfection. (G) Total resection of S’μ and S’α at S’μ-S’α junctions from blunt, 5’, and 3’DSBs in Ramos cells. Dotted line denotes the total overhang length in S’μ plus S’α (101 nt). (H) Microhomology usage at S’μ-S’α junctions from blunt, 5’, and 3’DSBs in Ramos cells. Black line denotes mean microhomology usage. ^The vast majority of S’μ-S’α junctions from blunt DSBs that were sequenced exhibited 0 nt of resection and 0 nt of microhomology, but had to be excluded from analysis for being non-unique.