Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

By Ayal Hendel, R. Bak, Joseph Clark, Andrew B. Kennedy, D. E. Ryan, Subhadeep Roy, Israel Steinfeld, Benjamin D Lunstad, R. Kaiser, Alec B. Wilkens and 5 more. Nature Biotechnology, 2015

1,033 citations recorded by the source corpus.

CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/a14f62c61ee1cfe50e860a59a1f15a1f53512683

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Citations in the corpus

• Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery. (2019, neutral): “Co-delivery Co-delivery of Cas9 mRNA and sgRNA or ZFN mRNA for ex vivo gene editing can be achieved via physical methods that bypass the extracellular and cytoplasmic barriers to deposit the molecular cargoes, such as electroporation 145,146 or microinjection into embryos.”
• mRNA-based therapeutics: powerful and versatile tools to combat diseases (2022, background): “466 The main consideration is about the efficiency, specificity, and safety of engineering T lymphocytes via mRNA transfection, chemically modified sgRNAs and Cas9 mRNAs increased genome editing efficiency via electroporation into human primary T cells in vitro.(467) Moreover, the delivery of Cas9 m”
• Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects (2020, background): “Similarly, Hendel et al.(146) recently demonstrated that the codelivery of chemically modified CCR5 sgRNA with Cas9 mRNA/protein enhanced the genome editing efficiency of human primary CD4+ T cells and CD34+ HSPCs, with no DNA delivery-associated toxicity.”
• Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides (2017, background): “5’ non-G nucleotides could be created via tRNA fusion [14] or chemical synthesis [15] and combined”