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 (Jinek et al., 2013), 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.
Highlights
The CRISPR-associated enzyme Cas9 enables site-specific genome engineering by introducing doublestrand breaks (DSB) at guide RNA-specified chromosomal loci of interest (Cong et al, 2013; Jinek et al, 2013; Mali et al, 2013a)
To test whether S phase is optimal for homology-directed repair (HDR) in HEK293T cells, six reversible chemical inhibitors were used in parallel experiments to synchronize HEK293T cells at G1, S and M phases of the cell cycle, followed by release prior to nucleofection with Cas9 RNP (Figure 1B, Figure 1—figure supplement 1A)
After 24 hr, cells were analyzed for HDR or total editing (TE, defined as the sum of all non-homologous end joining (NHEJ) and HDR events that give rise to indels) at the Cas9 cleavage site within EMX1, showing that both aphidicolin and nocodazole led to pronounced increases in Cas9-mediated editing frequencies (Figure 1D,E)
Summary
The CRISPR-associated enzyme Cas enables site-specific genome engineering by introducing doublestrand breaks (DSB) at guide RNA-specified chromosomal loci of interest (Cong et al, 2013; Jinek et al, 2013; Mali et al, 2013a). Cells repair DSBs using the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Cells have differing abilities to repair DSBs using NHEJ or HDR, the phase of the cell cycle largely governs the choice of pathway. NHEJ dominates DNA repair during G1, S and G2 phases, whereas HDR is restricted to late S and G2 phases when DNA replication is completed and sister chromatids are available to serve as repair templates (Heyer et al, 2010). Impediments to HDR include competition with NHEJ in S and G2 phases and specific down-regulation of HDR at M phase and early G1 to prevent telomere fusion (Orthwein et al, 2014). Chemical or genetic interruption of the NHEJ pathway can favor HDR (Shrivastav et al, 2008), such manipulations can be difficult to employ, harmful to cells or both. High cleavage activity of programmable nucleases does not necessarily correlate with efficient HDR-induced genome editing
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