Abstract
Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.
Highlights
Analysis of off-target editing is an important aspect of the development of safe nucleasebased genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome
We first tested whether biotin-dsDNA donors enable targeted nonhomologous end joining (NHEJ)-mediated insertion in an easy-to-transfect mouse neuroblastoma cell line, Neuro 2A (N2A)
In the presence of SpyCas[9], we observed a higher fraction of GFP+ cells for both dsDNA (6.9 ± 0.3%) and biotin-dsDNA (6.2 ± 0.2%), demonstrating that biotinylated dsDNA generated by PCR is competent for homology-independent insertion in cells
Summary
Analysis of off-target editing is an important aspect of the development of safe nucleasebased genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. In vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. The GUIDE-tag system utilizes tethering between the Cas[9] nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. Several methods for recruiting the DNA template to the Cas9-sgRNA complex to enhance homology-directed repair (HDR) have been reported in cells or mouse zygotes: (1) high-affinity biotin-streptavidin interactions[34], (2) direct or indirect covalent tethering, or (3) chemical modification[35,36,37,38,39]
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