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

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.

Figure 1.

Targeted mutagenesis in human K562 cells via direct delivery of RGEN RNPs. (A) CCR5-specific RGEN RNP-mediated mutations measured by the T7E1 assay. (B) Mutant DNA sequences at the CCR5 locus. The 20-bp target sequence is underlined and shown in bold. The PAM sequence is shown in red. (C) RGEN RNP-mediated mutagenesis at several endogenous loci. A mixture of Cas9 protein (15 μg) and sgRNA (20 μg) was transfected into 2 × 10⁵ K562 cells. PCR amplicons around RGEN target sites were subjected to the T7E1 assay. Representative data from at least three independent experiments are shown.

Figure 2.

Genome editing in BJ fibroblasts and H9 hES cell lines via direct delivery of RGEN RNPs. (A) CCR5-specific RGEN-driven mutations detected by the T7E1 assay in H9 and BJ cells. (B) RGEN-driven mutations in H9 ES cells detected by the T7E1 assay. A mixture of Cas9 protein (75 μg) and sgRNA (100 μg) was transfected into 1 × 10⁶ H9 cells. (C) Cytotoxicity of RGEN RNPs vs. RGEN plasmid in H9 ES cells. (**) P < 0.01, (*) P < 0.05. (D) No apparent changes in the physiology of ES cells after RGEN RNP treatment. Untransfected, RNP-, and plasmid-transfected ES cell colonies were subjected to AP staining.

Figure 3.

Homology-directed repair using ssODNs. The 86-mer ssODN includes an XbaI restriction enzyme site, which is absent at the target site, between two short homology arms. PCR amplicons were digested with XbaI in an RFLP assay to detect sequences that resulted from homology-directed repair.

Figure 4.

Targeted chromosomal deletions via RGEN RNPs. (A) RGEN target sites in the region of the CCR5 locus. The distances between the CCR5 site and each of the other sites are shown. Arrows indicate PCR primers. Red arrowheads indicate sgRNA target sites. (B) PCR products corresponding to deletions in K562 cells treated with RGEN RNPs; 15 μg of Cas9 protein premixed with 20 μg each of two sgRNAs was transfected into 2 × 10⁵ K562 cells. (C) DNA sequences of deletion-specific PCR products. In cases in which a sequence was detected more than once, the number of occurrences is shown in parentheses.

Figure 5.

Off-target mutations caused by RGEN RNPs vs. RGEN plasmids. RGEN RNPs or plasmids that encode Cas9 and sgRNA were electroporated into K562 cells. Mutations were detected using the T7E1 assay (left) and deep sequencing (right). The PAM sequence is shown in blue. Mismatched bases are shown in red.

Figure 6.

Time-course analyses of RGEN-mediated genome editing via RNP delivery or plasmid transfection. (A, top) Mutation frequencies were determined by the T7E1 assay. (Bottom) Western blot analysis of K562 cells transfected with the CCR5-specific RGEN via RNP or plasmid DNA delivery. (B,C) Line graphs showing the results of the T7E1 (B) and Western blot analysis (C). Note that only the relative abundance of Cas9 in each experiment is shown.