American Society of Hematology

Figure 6.

$Identification of Cas9 off-target sites by GUIDE-seq and quantification of potential Cas9 off-target cleavage sites using rhAmpSeq technology. (A) Sequences of off-target sites identified by GUIDE-seq for 2 guides targeting the CISH locus. The guide sequence is listed on top with off-target sites shown below. The on-target site is identified with a black square. Mismatches to the guide are shown and highlighted in color with insertions shown in gray. The number of GUIDE-seq sequencing reads are shown to the right of each site. 10 µM Alt-R crRNA XT complexed to Alt-R transactivating CRISPR RNA was delivered into HEK293 cells that constitutively express Cas9 nuclease by nucleofection. (B) Pie charts indicate the fractional percentage of the total unique, CRISPR-Cas9–specific read counts that are on-target (red) and off-target (blue). Total editing at the on- and off-target sites identified by GUIDE-Seq was measured using rhAmpSeq, a multiplexed targeted enrichment approach for next-generation sequencing. For each of the 2 CISH targeting guides, amplicons were designed around each Cas9 cleavage site with reads >1% of the on-target GUIDE-seq reads. RNP complexes formed with either WT Cas9 (blue) or Alt-R HiFi Cas9 (red) were delivered via electroporation into expanded NK cells. (C) Insertion/deletion formation at each targeted loci for CISH guide 1 (panel 1, 11-plex) and CISH guide 2 (panel 2, 70-plex) when a single RNP complex was delivered. The on-target locus is indicated with a black asterisk underneath the first 2 bars of each graph. (D) Insertion/deletion formation at each targeted loci when CISH guide 1 and CISH guide 2 were codelivered. The on-target locus is indicated with a black asterisk underneath the first 2 bars of each graph.$

Identification of Cas9 off-target sites by GUIDE-seq and quantification of potential Cas9 off-target cleavage sites using rhAmpSeq technology. (A) Sequences of off-target sites identified by GUIDE-seq for 2 guides targeting the CISH locus. The guide sequence is listed on top with off-target sites shown below. The on-target site is identified with a black square. Mismatches to the guide are shown and highlighted in color with insertions shown in gray. The number of GUIDE-seq sequencing reads are shown to the right of each site. 10 µM Alt-R crRNA XT complexed to Alt-R transactivating CRISPR RNA was delivered into HEK293 cells that constitutively express Cas9 nuclease by nucleofection. (B) Pie charts indicate the fractional percentage of the total unique, CRISPR-Cas9–specific read counts that are on-target (red) and off-target (blue). Total editing at the on- and off-target sites identified by GUIDE-Seq was measured using rhAmpSeq, a multiplexed targeted enrichment approach for next-generation sequencing. For each of the 2 CISH targeting guides, amplicons were designed around each Cas9 cleavage site with reads >1% of the on-target GUIDE-seq reads. RNP complexes formed with either WT Cas9 (blue) or Alt-R HiFi Cas9 (red) were delivered via electroporation into expanded NK cells. (C) Insertion/deletion formation at each targeted loci for CISH guide 1 (panel 1, 11-plex) and CISH guide 2 (panel 2, 70-plex) when a single RNP complex was delivered. The on-target locus is indicated with a black asterisk underneath the first 2 bars of each graph. (D) Insertion/deletion formation at each targeted loci when CISH guide 1 and CISH guide 2 were codelivered. The on-target locus is indicated with a black asterisk underneath the first 2 bars of each graph.

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