Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components

Ignazio Maggio,Hidde A Zittersteijn,Manuel A F V Gonçalves,Ivan Toral Ojeda,Silvère M Van Der Maarel,Qian Wang,Jin Liu,Rob C Hoeben,Arjan C Lankester,Josephine M Janssen

doi:10.1038/s41434-019-0119-y

Abstract

Enhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.

Highlights

RNA-guided nucleases (RGNs) derived from prokaryotic adaptive immune systems are being repurposed for genome editing applications
We investigated the effects on nuclear localization and targeted DSB frequencies of RGNs containing the high-specificity eSpCas9(1.1) nuclease with two or four nuclear localization signals (NLSs)
The Cas9 construct contains a human codon-optimized Streptococcus pyogenes Cas9 (SpCas9) open reading frame (ORF) linked to a SV40 large T antigen (SV40) NLS sequence [5], whereas the eCas9.2NLS construct contains the eSpCas9 (1.1) ORF flanked by the SV40 and nucleoplasmin NLS sequences [17] (Fig. 1a)

Summary

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RNA-guided nucleases (RGNs) derived from prokaryotic adaptive immune systems are being repurposed for genome editing applications. The amino acids substitutions N497A/R661A/ Q695A/Q926A and N692A/M694A/Q695A/H698A have generated the high-specificity SpCas9-HF1 and HypaCas variants, respectively [15, 16] These mutations are localized within REC3, a noncatalytic domain of SpCas involved in RNA–DNA heteroduplex recognition and in conformational activation of the HNH nuclease domain [15, 16]. Despite constant efforts to optimize CRISPR-Cas components, further improvements on intracellular delivery and target DNA cleavage remain in demand for advancing genome editing in human cells [2, 23]. To address these requirements, we assembled RGNs containing optimized SpCas and gRNA components. In contrast to other recombinant viral systems, e.g., lentiviral and adenoassociated viral vectors, AdVs have a strict episomal nature and large packaging capacity permitting high-level transitory expression of sizable RGN-encoding transgenes, both in vitro and in vivo [26,27,28,29,30]

Results

Discussion

Materials and methods

Compliance with ethical standards