Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

TLDR

CRISPR/Cas9 is a robust genome editing technology that cleaves DNA in a RNA‑programmed manner in human cells, animals, and plants. The study aims to show that timed delivery of Cas9‑guide RNA ribonucleoprotein complexes can introduce new genetic information site‑specifically and efficiently via homology‑directed repair. This is accomplished by delivering Cas9‑guide RNA ribonucleoprotein complexes at a controlled time to induce site‑specific double‑strand breaks for HDR. HDR rates increased dramatically, reaching up to 38 % in HEK293T cells, with high‑fidelity editing and minimal cell mortality, demonstrating a simple and highly effective strategy for enhancing site‑specific genome engineering in transformed and primary human cells.

Abstract

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (<xref ref-type="bibr" rid="bib13">Jinek et al., 2013</xref>), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.