Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

Abstract

CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

Figure 1

Synthesized and chemically modified sgRNAs facilitate high frequencies of indels and HR in the human cell line K562. (a) Sequence and schematic secondary structure of the IL2RG sgRNA loaded into Cas9 and bound to its genomic target site. Nucleotides with chemical modifications are marked with red flags. (b) Structures of chemical modifications incorporated during chemical synthesis of sgRNAs (sequences can be found in Supplementary Table 1). (c,d amplicons (c) or gene addition by HR at the three loci IL2RG, HBB and CCR5 with synthetic sgRNAs (d). The synthetic sgRNAs were delivered at 1 μg (light shade) or 20 μg (dark shade) per 1 million cells. Cas9 was expressed from a plasmid (2 μg) and for HR experiments 5 μg of GFP-encoding donor plasmid was included. As a positive control, 2 μg of sgRNA plasmid encoding both the sgRNA and the Cas9 protein was used (gray bars). Bars represent average values + s.e.m., n = 3. (e) Specificity of targeted cleavage mediated by synthetic sgRNAs as performed in c for 20 μg of sgRNA. Indel frequencies were measured by deep sequencing of PCR amplicons of the targeted genomic loci and three bioinformatically predicted off-target loci for each gene. Bars represent average values + s.e.m., n = 3. (f) Staggered delivery of 15 μg Cas9 mRNA and 10 μg IL2RG synthetic sgRNAs into 1 million K562 cells using electroporation. Bars represent average indel frequencies + s.e.m., n = 3, as measured by tracking of indels by decomposition (TIDE) analysis of PCR amplicons spanning the sgRNA target sites, using a mock-treated sample as reference control. (g) Cas9 protein was complexed with a 2.5 molar excess of the indicated synthetic IL2RG sgRNAs and nucleofected into 1 million K562 cells at the indicated amounts. Indel frequencies were measured by TIDE analysis as above and bars represent average indel frequencies + s.e.m., n = 3.

Figure 2

Chemically modified sgRNAs facilitate high frequencies of gene disruption in stimulated primary human T cells and CD34⁺ hematopoietic stem and progenitor cells (HSPCs). (a) 1 million primary human T cells were nucleofected with 10 μg of the indicated synthetic CCR5 sgRNAs and either 15 μg Cas9 mRNA or 1 μg Cas9-encoding plasmid. 1 μg sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies for three different donors + s.e.m., n = 6, as measured by TIDE analysis of PCR amplicons spanning the sgRNA target site, and using a mock-treated sample as control reference. (b) Stimulated T cells were nucleofected as above, but with 15 μg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs. Indel frequencies were measured by TIDE analysis as above. Bars represent average indel frequencies for three different donors + s.e.m., n = 6. (c) 500,000 mobilized human peripheral blood CD34⁺ HSPCs were nucleofected with 10 μg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 μg Cas9 mRNA or 1 μg Cas9 plasmid. 1 μg of sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies + s.e.m., n = 3, as measured by T7 endonuclease cleavage assay. (d) 1 million stimulated T cells or mobilized human peripheral blood CD34⁺ HSPCs were nucleofected with 15 μg Cas9 mRNA and 10 μg of the indicated synthetic CCR5 sgRNAs. When used in combination the amount of each sgRNA was 5 μg. Indel frequencies for samples with single sgRNAs were measured by TIDE analysis as above and allele disruption frequencies for samples with two sgRNAs were measured by sequencing of cloned PCR products (Supplementary Fig. 15). Bars represent average indel frequencies + s.e.m., n = 3.