Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

By Sojung Kim, Daesik Kim, S. Cho, Jungeun Kim, Jin-Soo Kim. Genome Research, 2014

1,716 citations recorded by the source corpus.

RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/20f2741892d4aeb930030284f9537d27dc5a0f61

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In Molecular Biology

Citations in the corpus

Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA[OPEN] (2015, background): “Furthermore, transient delivery of purified Cas9 protein and gRNA (as a ribonucleoprotein complex) into human culture cells has shown high frequencies of targeted mutagenesis and gene editing while reducing off-target mutations (Kim et al., 2014; Lin et al., 2014).”
Target specificity of the CRISPR-Cas9 system (2014, neutral): “transcribed sgRNA, either individually or as purified complex, reduces off-targets in cells [66,67].”
Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery (2014, background): “Previous reports have shown ∼20% NHEJ with transfection of Cas9 RNPs but didn't analyze HDR rates (Kim et al., 2014).”
Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA (2014, background): “In addition, a similar study in review at the same time reported highly efficient genome editing through electroporation-mediated delivery of purified (unmodified) Cas9 protein and sgRNA complex into human cells (Kim et al. 2014).”
Engineering the Delivery System for CRISPR-based Genome Editing (2018, background): “Delivery by encapsulation Lipid-based carriers are a popular and efficient delivery system for CRISPR DNA, mRNA, or RNPs. Lipids are naturally occurring amphiphilic organic molecules composed of a hydrophilic head group and a long-chain hydrophobic tail group.”
Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides (2017, background): “Delivery of pre-assembled Cas9 variant ribonucleoproteins [16] rather than Cas9- and sgRNA-encoding plasmids may”