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

Authors:
Kim S, Kim D, Cho SW, Kim J, Kim JS
In:
Source: Genome Res
Publication Date: (2014)
Issue: 24(6): 1012-9
Research Area:
Cancer Research/Cell Biology
Stem Cells
Basic Research
Molecular Biology
Cells used in publication:
K-562
Species: human
Tissue Origin: blood
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
BJ
Species: human
Tissue Origin: dermal
H9
Species: human
Tissue Origin: blood
Platform:
4D-Nucleofector™ X-Unit
Experiment

To introduce DSBs in mammalian cells using an RNP complex, 2E+5 cells were transfected with Cas9 protein (4.5–45 µg) premixed with in vitro transcribed sgRNA (6–60 µg). Cas9 protein in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol) was mixed with sgRNA dissolved in nuclease-free water and incubated for 10 min at room temperature. No more than 4 µL of the RNP mixture was added to 20 mL of the Nucleofection solution. For plasmid-mediated expression of RGENs, 2E+5 cells were co-transfected with 1 mg of Cas9-encoding plasmid and 1 µg of sgRNA-expressing plasmid in K562 and BJ fibroblasts or 2.4 µg of Cas9-encoding plasmid and 1.6 µg of sgRNA-expressing plasmid in H9 hES cells. K562 cells were transfected with the Amaxa SF Cell Line 4D-Nucleofector Kit using Program FF-120 (Lonza), and H9 and BJ cells were transfected with the Amaxa P3 Primary Cell 4D Nucleofector Kit using Program CB-150 and DT-130, respectively, according to the manufacturer’s protocol. Cells were analyzed 2 days after transfection, unless indicated otherwise. Lonza summary: The authors delivered purified recombinant Cas9 protein and guide RNA, from CRISPR system, into K562, human fibroblasts and pluripotent stem cells using the 4D Nucleofector system. This reduces off-target mutations and was less stressful for human embryonic stem cells, in comparison to Cas9 plasmid transfection, due to the rapid degradation of the transfected Cas9 ribonucleoprotein after delivery. The mutation frequency reached a plateau one day upon Nucleofection when Cas9 was transfected as a protein versus three days when it was delivered as a plasmid. The versatility of Nucleofection for delivering different type of substrates including large proteins without further Nucleofection optimization enabled here the improvement of genome engineering methods.

Abstract

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.