Abstract
Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing reagents, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins with a nucleic acid donor template, as well as other biotinylated molecules such as quantum dots. In human cells, tailored S1mplexes increase the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene-editing methods, and enrich cell populations containing multiplexed precise edits up to 42-fold. These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro or ex vivo gene-editing applications in precision medicine and drug discovery and aid in the development of increased and serial dosing regimens for somatic gene editing in vivo.
Highlights
Writing specific DNA sequences into the human genome is challenging with non-viral geneediting reagents, since most of the edited sequences contain various imprecise insertions or deletions
This break is predominately repaired through one of two major DNA repair pathways: error prone non-homologous end-joining (NHEJ) or precise homology directed repair (HDR), in which a template is used for precise gene editing
Variable delivery of the CRISPR system along with the donor templates generates a spectrum of edits, where a majority of cells include imprecise insertions and deletions of DNA bases from NHEJ or microhomology mediated end-joining of the DSB7, 8
Summary
Writing specific DNA sequences into the human genome is challenging with non-viral geneediting reagents, since most of the edited sequences contain various imprecise insertions or deletions. (SpCas[9], a high-affinity nuclease isolated from a type II CRISPR–associated system) and a single-guide RNA (sgRNA) generate on-target DNA double-strand breaks (DSBs) with little to no off-target DNA cleavage[5, 6] This break is predominately repaired through one of two major DNA repair pathways: error prone non-homologous end-joining (NHEJ) or precise homology directed repair (HDR), in which a template is used for precise gene editing. Selection strategies through viral integration and excision of drug[16] or cell-surface[1] selection cassettes, flow cytometry for co-expressed fluorescent proteins[10, 17, 18], or through transient drug selection (Steyer B.G. et al, submitted) can assist in the isolation of cells with one or two precisely edited alleles[7] For all of these strategies, imprecise editing through NHEJ typically outnumbers precise HDR outcomes[2, 6]. We reasoned that some of the noise in gene-editing outcomes could be reduced by preassembling RNPs with the donor template or other moieties that enable the isolation of precisely edited cells
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