Fig. 1

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 < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. O original, M mutant