Extended Data Fig. 4: Examination of off-target integrations with non-viral genome targeting.

a, Results of targeted locus amplification (TLA) sequencing. No off-target integration sites were identified (assay's limit of detection ~1% of alleles) with either a dsDNA or ssDNA HDR template in two healthy donors. The on-target RAB11A locus on chromosome 15 is indicated in red. b, The frequency of one of the observed incorrect integrations at the target locus was reduced using a long ssDNA HDR template in two human blood donors (Supplementary Note 2). c, Diagram of HDR-mediated insertions at the N terminus of a target locus. The homology arms specify the exact sequence where the insert (a GFP tag in this case) will be inserted, allowing for scarless integration of exogenous sequences. Because a GFP fusion protein is created, GFP fluorescence will be seen as a result of the on-target integration, which is dependent on an RNP cutting adjacent to the integration site. d, dsDNA can be integrated via homology-independent repair mechanisms at off-target sites through either random integration at naturally occurring dsDNA breaks, or potentially at induced double-stranded breaks, such as those at the off-target cut sites of the RNP. This effect can be harnessed to allow for targeted integration of a dsDNA sequence at a desired induced dsDNA break in quiescent cell types which lack the ability to do HDR, but crucially the entire sequence of the dsDNA template is integrated, including any homology arms. In the case that the homology arms contain a promoter sequence (such as for N-terminal fusion tags), these off target integrations can drive observable expression of the inserted sequence without the desired correct HDR insertion. e, We looked for unintended non-homologous integrations with the non-viral system using an N-terminal GFP-RAB11A fusion construct that contained the endogenous RAB11A promoter sequence within its 5′ homology arm. This construct could express GFP at off-target integration sites, which allowed us to assay for off-target events at the single-cell level using flow cytometry. Inclusion of a gRNA designed to cut a genome region that is not the homologous region to the targeting sequence can be used to infer integration at an off-target cut site. f, Although efficient GFP expression depended on pairing the HDR template with the correct gRNA targeting that site, rare GFP⁺ cells were observed when dsDNA HDR templates were delivered either alone (~0.1%) or with an off-target Cas9 RNP (~1%). g, Quantification of different types of functional off-target integrations. The increase in the percentage of fluorescent cells over the limit of detection when the template alone is electroporated probably represents random integrations at naturally occurring dsDNA breaks (although cut-independent integration at the homology site is also possible in theory). Not every off-target integration will yield fluorescent protein expression (for example, only part of the template sequence could be integrated or it could be integrated in a way that does not lead to measurable expression), but the relative differences in functional off-target expression between different templates and editing conditions can be assayed. Inclusion of an RNP targeting CXCR4 (off-target) markedly increased the observed off-target homology-independent integrations, probably by a homology-independent insertion event. As expected, efficient GFP expression as expected was only seen with the correct gRNA sequence and HDR-mediated repair. Bars represent observed GFP⁺ percentages from T cells from one representative donor electroporated with the indicated components. h, Comparisons of on-target GFP expression versus functional off-target integrations across five templates reveal HDR is highly specific, but that off-target integrations can be observed at low frequencies. i, A matrix of gRNAs and HDR templates were electroporated into bulk T cells from two healthy donors. The average GFP expression in gated CD4⁺ T cells as a percentage of the maximum observed for a given template is displayed. Across six unique HDR templates and gRNAs, on-target HDR-mediated integration was the by far most efficient. One HDR template, a C-terminal GFP fusion tag into the nuclear factor FBL, had consistently higher off-target expression across gRNAs, potentially due to a gene-trap effect as the 3′ homology arm for FBL contains a splice-site acceptor followed by the final exon of FBL leading into the GFP fusion. n = 2 (a, b, h, i) or n = 8 (e, f) independent healthy donors.