Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

Yvonne Baron,Johanna Sens,Lucas Lange,Larissa Nassauer,Denise Klatt,Dirk Hoffmann,Marc-Jens Kleppa,Philippe Vollmer Barbosa,Maximilian Keisker,Viviane Steinberg,Julia D Suerth,Florian W.R Vondran,Johann Meyer,Michael Morgan,Axel Schambach,Melanie Galla

doi:10.1016/j.omtn.2021.12.033

Yvonne Baron, Johanna Sens + Show 14 more

Open Access

https://doi.org/10.1016/j.omtn.2021.12.033

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Abstract

DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous TP53/Trp53 genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34+ stem and progenitor cells. Strikingly, co-packaging of Cas9 mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.

Highlights

Like other DNA-modifying enzymes, CRISPR-Cas[9] has limitations and should be expressed in a dose-controlled manner, as constitutive expression of CRISPR-Cas[9] components was shown to result in off-target events and to inhibit cell proliferation, even in the absence of a co-expressed single guide RNA.[16]
The concept of g.group-specific antigen (Gag).MS2.CRISPR particles The recently described g.Gag.MS2.CRISPR particles were designed for spatiotemporal co-delivery of Cas[9] and single guide RNA (sgRNA) transcripts into target cells
The preservation of most Gag subdomains should allow the generation of retrovirus-like particles, which protect the nonretroviral RNA cargo from extracellular nucleases and facilitate, together with the envelope glycoprotein, introduction of the RNA cargo into cells in a non-toxic manner

Summary

Introduction

Like other DNA-modifying enzymes, CRISPR-Cas[9] has limitations and should be expressed in a dose-controlled manner, as constitutive expression of CRISPR-Cas[9] components was shown to result in off-target events and to inhibit cell proliferation, even in the absence of a co-expressed single guide RNA (sgRNA).[16]. One possibility is the use of non-integrating reverse transcription disabled retroviral vectors,[29,30,31,32,33,34] as they—like their integrating wild-type (wt) counterparts— possess all retroviral properties for efficient and non-toxic cell entry but can be retargeted to any desired cell type by innovative pseudotyping strategies.[35,36,37,38] generated alpharetroviral Gag.MS2 (a.Gag.MS2) particles with g.Gag.MS2 particles revealed enhanced CRISPR-Cas[9] delivery into target cells These particles allowed efficient targeted knockout of endogenous TP53/Trp[53] genes in several primary cell sources. We report optimized a.Gag.MS2 particles as a versatile tool for short-term and dose-controlled delivery of CRISPR-Cas[9] into target cells

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