Journal: Nature biotechnology
Article Title: Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells
doi: 10.1038/nbt.3290
Figure Lengend Snippet: 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.
Article Snippet: Cell culture and nucleofections K562 (ATCC) and T cells were cultured at 37 °C, 5% CO 2 , and ambient oxygen levels.
Techniques: Synthesized, Modification, Sequencing, Plasmid Preparation, Positive Control, Electroporation, Control
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