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. 2015 Dec 15;16:280.
doi: 10.1186/s13059-015-0846-3.

Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency

Ying Dang  1 , Gengxiang Jia  2 , Jennie Choi  3 , Hongming Ma  4 , Edgar Anaya  5 , Chunting Ye  6 , Premlata Shankar  7 , Haoquan Wu  8
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Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency

Ying Dang et al. Genome Biol. .
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Abstract

Background: Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)-transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency.

Results: Here, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the optimized sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss of function in non-coding genes.

Conclusions: By a systematic investigation of sgRNA structure we find that extending the duplex by approximately 5 bp combined with mutating the continuous sequence of thymines at position 4 to cytosine or guanine significantly increases gene knockout efficiency in CRISPR-Cas9-based genome editing experiments.

Figures

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 P P P O original, M mutant
Fig. 2
Knockout efficiency can be further increased by combining duplex extension with disruption of the continuous sequence of Ts. a The effect of duplex extension when mutating the fourth T to an A in four sgRNAs. The raw data are shown in Figure S4 in Additional file 1. b The effect of mutation of Ts at the indicated positions to A, C, or G when also extending the duplex by 5 bp. The raw data are shown in Figure S5 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 P P P M mutant
Fig. 3
The optimized sgRNA structure is superior to the original version. a CCR5 knockout efficiency was determined for the indicated sgRNAs targeting CCR5 with either an optimized sgRNA structure or the original structure. The knockout efficiency was determined in the same way as in Fig. 1b. The raw data are sown in Figure S6 in Additional file 1. b CD4 knockout efficiency was determined for the indicated sgRNAs targeting the CD4 gene, with two versions of the sgRNA structure in Jurkat cells. Cells were analyzed for CD4 expression by flow cytometry 72 hours after transfection. The raw data are shown in Figure S7 in Additional file 1. c T→C and T→G mutations are superior to the T→A mutation. Eleven sgRNAs targeting CCR5 were randomly selected. The knockout efficiency of sgRNAs with different mutations at position 4 in the sequence of continuous Ts were determined as in Fig. 1c. The raw data are shown in Figure S9 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 P P P 
Fig. 4
The efficiency of gene deletion is increased dramatically using optimized sgRNAs. a The CCR5 gene deletion. b sgRNA pairs targeting CCR5 with the original or optimized structures were co-transfected into TZM-bl cells with a Cas9-expressing plasmid. The gene deletion efficiency was determined by amplifying the CCR5 gene fragment. Note that the truncated fragments of CCR5, with a smaller size than wild-type CCR5, are a consequence of gene deletion using paired sgRNAs. The numbers below each lane indicate the percentage deletion
Fig. 5
How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b. The raw data are shown in Figure S10 in Additional file 1. Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small RNA. c In vitro transcribed sgRNA formed dimers (upper panel), which can be transformed into monomers by a heating and quick cooling step (lower panel). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b. The raw data are shown in Figure S11 in Additional file 1. e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b. The raw data are shown in Figure S11 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 P 
Fig. 6
Testing the effect of modifications by lentiviral infection. TZM-bl cells (a) or JLTRG-R5 cells (b) were infected with Cas9-expressing lentivirus, and cells stably expressing Cas9 were selected. The indicated sgRNA (sp3 from Fig. 2a)-expressing cassettes were packaged into lentivirus and used to infect cells stably expressing Cas9 at MOI = 0.5. Knockout efficiency was determined as in Fig. 1b on the indicated days. The raw data are shown in Figure S12 in Additional file 1. O original, Mut mutant
Fig. 7
Optimized sgRNA structure. The duplex extension is highlighted in red, and the mutation is marked in bold. The duplex extension can be four to six nucleotides, and the mutation can be C or G, which showed similar knockout efficiency in most cases
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