Abstract
BackgroundThe application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications.ResultsWe found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion.ConclusionsOur results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.
Highlights
The application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9) technology in human induced pluripotent stem cells holds tremendous potential for basic research and cell-based gene therapy
Bacteriophage-chimeric retrovirus-like (LentiFlash®) particles for highly efficient and transient RNA delivery in human induced pluripotent stem cells (hiPSC) To investigate whether LentiFlash® particles can transduce hiPSC, we generated particles containing the fluorescent reporter ZsGreen mRNA (LF-ZsGreen) and used them at three doses (0.5, 2, and 5 pg p24/cell) to transduce a hiPSC line (HY03) derived from a healthy individual [23]
These findings confirmed previous data obtained in HeLa cells and clearly show that we can quantify ZsGreen RNA and ZsGreen protein expression in cells transduced with LentiFlash® particles
Summary
The application of CRISPR/Cas technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. Human pluripotent and induced stem cells (hPSC and hiPSC, respectively) can self-renew, display unlimited proliferative potential, and can differentiate into any cell type of the three germ layers (ectoderm, mesoderm, endoderm) They hold tremendous potential for basic research and disease modeling, and for cell production for clinical applications [1, 2]. This potential has been magnified by the development of gene editing tools, those based on clustered regularly interspaced short palindromic repeats (CRISPR) technologies, to genetically manipulate the hPSC genome [3, 4]. Despite recent improvements based on new strategies, such as the transfection of ribonucleoprotein complexes, and the development of new gene transfer tools to enhance delivery in hPSC [7,8,9,10,11,12,13,14], new systems are still required to efficiently deliver nucleic acids in hPSC with minimal toxicity
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