Journal: Genome Biology
Article Title: Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency
Figure Lengend Snippet: Knockout efficiency can be increased by extending the duplex and disrupting the continuous sequence of Ts. a The duplex extension. Green indicates the 3’ 34 nucleotides, which are not required for sgRNA functionality in vitro but are required in cells; red indicates the extended base pairs. b Extension of the duplex increased knockout efficiency. Constructs harboring sgRNAs targeting the CCR5 gene were co-transfected with a Cas9-expressing plasmid into TZM-bl cells. An sgRNA targeting the HIV genome served as mock control. The GFP-positive cells were sorted out 48 hours after transfection, and the gene modification rates were determined at the protein and DNA levels, respectively. Protein level disruption: the expression of CCR5 was determined by flow cytometry analysis. The raw data are shown in Figure S2 in Additional file 1 . DNA level modification rate: the genomic DNA was extracted, and the target sites were amplified and deep-sequenced with a MiSeq sequencer. The raw data are provided in Additional file 2 . c The experiment in ( b ) at the protein level was repeated for another sgRNA, sp2. The difference with ( b ) is that the cells were not sorted, but the CCR5 disruption rate was measured in GFP-positive cells. The raw data are shown in Figure S2 in Additional file 1 . d Mutation of the RNA polymerase ( Pol III ) pause signal significantly increased knockout efficiency. The mutated nucleotides are shown in bold . The raw data are shown in Figure S3 in Additional file 1 . The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
Article Snippet: One microgram of extracted RNA was reverse transcribed with SuperScript® III Reverse Transcriptase reaction (Life Technology, catalog #18080-051), according to the manufacturer’s instructions.
Techniques: Knock-Out, Sequencing, In Vitro, Construct, Transfection, Expressing, Plasmid Preparation, Modification, Flow Cytometry, Cytometry, Amplification, Mutagenesis, Standard Deviation