Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.
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.
Department of Chemistry, Seoul National University, Seoul 151-747, South Korea; Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea.
Sojung
S
Kim
Department of Chemistry, Seoul National University, Seoul 151-747, South Korea; Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea.
Daesik
D
Kim
Department of Chemistry, Seoul National University, Seoul 151-747, South Korea; Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea.
Seung Woo
SW
Cho
Department of Chemistry, Seoul National University, Seoul 151-747, South Korea; Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea.
Jungeun
J
Kim
Department of Chemistry, Seoul National University, Seoul 151-747, South Korea; Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea.
Jin-Soo
JS
Kim
D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D015894:Genome, Human; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods; D039904:Pluripotent Stem Cells; D017354:Point Mutation; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism
D000094704:RNA, Guide, CRISPR-Cas Systems; D012261:Ribonucleoproteins