Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing

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.

Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

“…Moreover, this strategy maintains the native sgRNA to Cas9 interaction and does not require the addition of other proteins, in contrast to recent studies 11 . By simply appending a 13 base pair recognition sequence to the 5′-end of an ssODN and using a Cas9-PCV fusion protein, HDR efficiency can be increased up to 30-fold as measured by three separate approaches.…”

“…In a recently described method termed S1mplex 11 , authors used recombinant streptavidin to bridge a modified single-guide RNA (sgRNA) containing a streptavidin-binding aptamer and a biotinylated single-stranded oligodeoxynucleotide (ssODN) to deliver a linked RNP-donor DNA complex and observed enhancement in precise genome editing compared to unlinked components. However, this method, along with other recent attempts to co-deliver the donor DNA using biotin-avidin 12 and the SNAP tag 13 , requires additional steps and expense associated with modifying the donor DNA and inclusion of additional recombinant proteins.…”

Transcription Factor–Mediated Differentiation of Human iPSCs into Neurons

“…Precise, label-free editing of endogenous loci may be enriched by FACS sorting for transient expression of the components necessary for editing 1 day after transfection [i.e., using a combined gRNA, Cas9, and GFP expression plasmid with an integration plasmid expressing RFP outside of the insertion sequence or by using functionalized RNPs such as S1mplex with Qdots or fluorescent streptavidin (Carlson-Stevermer et al, 2017)]. This can considerably increase the prevalence of correctly edited cells, but the combined stress of transfection and sorting can decrease cell survival.…”

Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing

“…In addition to repurposing responsive CRISPR/Cas9, combination of gRNA with aptamers that can recruit various protein has greatly broadened its possible applications [66], for example, simplified gene activation [67,68], enhanced gene knock-in [69] and orthogonal gene manipulation [70]. The promising aptamer-reprogrammed sgRNA has a potential to take the merit of numerous aptamers but also suffers from aptamers' background activity without binding ligands and limited dynamic range.…”