Partial DNA-guided Cas9 enables genome editing with reduced off-target activity

Abstract

CRISPR-Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9-sgRNA complex as a guide, the majority of the 3' end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3'-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA-RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells.

Figure 1. Partial DNA replacement at the guide region of a GFP crRNA induces gene editing in human cells

(a) Diagram of the CRISPR system. PAM, protospacer adjacent motif. (b) HEK293T cells stably expressing both elongation factor-1 short (EFs) promoter-SpCas9 and elongation factor 1-α (EF1α) promoter-GFP were transfected with a crRNA targeting GFP and the tracrRNA. Cas9-mediated frame-shift NHEJ yields GFP-negative cells. When replacement of DNA nucleotides in crRNA is tolerated by Cas9, the percent of GFP-negative cells will be retained. The blue arrowhead indicates the cutting site by the Cas9. (c) Illustration of DNA replacement at the guide sequence of GFP crRNAs. The 20-nucleotide (nt) guide region is shown. RNA and DNA are shown in black and red, respectively. (d) HEK293T cells described above were incubated with the tracrRNA and a GFP-targeting crRNA illustrated. TIDE analysis was performed to calculate the percent of indels at GFP locus at day 3. n = 9 biologically independent samples. Error bars show mean ± s.d. Purple color indicates mock-transfection-treated samples. Black dots indicate native crRNA transfected samples. Red dots indicate DNA–RNA chimeric crRNAs-transfected samples.

Figure 2. Partial DNA replacement at the guide region of crRNA or sgRNA induces efficient gene editing in human cells

(a) Partial DNA replacement at the guide region of a crRNA targeting EMX1 induced indels in human cells. HEK293T cells described above were transfected with the tracrRNA and an EMX1-targeting crRNA. TIDE analysis was performed to determine indels at EMX1 locus. n = 3 biologically independent samples. *P < 0.01 by one-way ANOV A with Tukey post hoc test. (b) sgRNAs targeting GFP, EMX1 or VE GFA with 8-nt DNA and a 10-nt replacement at the 5′ end (sgRNA-8 DNA and sgRNA-10 DNA) induced indels in HEK293T cells. HEK293T cells described above were transfected with a native sgRNA or sgRNA-8D or sgRNA-10D. TIDE analysis was performed to determine percent of indels. n = 3 biologically independent samples. Black and red dots indicate native crRNA and DNA–RNA chimeric crRNAs transfected samples, respectively. (c) DNA–RNA chimeric crRNAs or sgRNA can mediate efficient genome editing in a RNP setting. VEGFA crRNA + tracrRNA or sgRNA alone were incubated with SpCas9 protein to form RNP complexes and then electroporated into Jurkat T cells by Neon transfection. Genomic DNA was harvested at day 3, and percent of indels was measured by TIDE. n = 3 biologically independent samples. (d) A DNA–RNA chimeric crRNA guides AsCpf1 for efficient genome editing. HEK293T cells were co-transfected with a plasmid expressing AsCpf1 and a native crRNA or crRNA with 8-nt DNA replacement at the 3′ end targeting DNMT1. TIDE analysis was performed to determine percent of indels at the DNMT1 locus 3 d after transfection. n = 3 biologically independent samples. (e,f) 4-nt DNA replacement at the 3′ end of the guide sequence (red), 4-nt mutation of DNA (DNA mut-1 or 2, pink) at the 5′ end, or 4-nt mutation of RNA (RNA mut, cyan) abolished activity of CRISPR in human cells. (e) Illustration of DNA replacement at the 3′ end and DNA or RNAmutations. (f) HEK293T cells described above were incubated with the tracrRNA and a GFP-targeting crRNA as in e. *P < 0.01; n = 6 biologically independent samples. Error bars represent mean ± s.d.

Figure 3. Partial DNA replacement at the guide region reduces off-target effects in human cells

(a) Illustration of DNA replacement at the guide sequence of VEGFA crRNA. Arrows denote mismatches between target and off-target sites. (b,c) HEK293T cells described in Figure 1 were transfected with the tracrRNA and a VEGFA-targeting crRNA with 10 DNA nt replacement at the 5′ end of the guide sequence (10 DNA) or with native crRNA. Surveyor assay were performed to determine indels at the VEGFA locus (b) and 3 top off-target (OT) sites of the VEGFA guide sequence (c). n = 3 biologically independent samples. *P < 0.01 by one-way ANOV A with Tukey post hoc test. (d,e) Partial DNA replacement in GFP crRNA reduces off-target activity. (d) Illustration of mismatch mutations of GFP2 sequences. Arrows denote point mutations. (e) TIDE analysis was performed to determine percent of indels. n = 3–5 biologically independent samples, as indicated. *P < 0.05 by unpaired, two-tailed Student’s t-tests. (f) GUIDE-seq genome-wide off-target analysis of native and 10 DNA crRNAs from three endogenous genes. The chart indicates the number of off-target peaks detected by GUIDE-seq for each type of crRNA. Six total mismatches are allowed in the guide and PAM. (g) Number of GUIDE-seq reads of 293 site 4. Target is the crRNA target site. OT 1–OT 6 are top off-target sites in the native crRNA data set. Error bars represent mean ± s.d.

Figure 4. An optimized DNA–RNA chimeric crRNA enables efficient genome editing in human cells

(a) Illustration of DNA substitution of GFP targeting crRNAs. RNA and DNA are shown in black and red, respectively. The Cas9 binding region is shown by a blue box. (b) U2OS-GFP-PEST cells stably expressing Cas9 were transfected with GFP crRNAs and the tracrRNA. GFP negative cells caused by Cas9-mediated frame shift NHEJ were measured by FACS at day 3. 8 DNA16 DNA design (8-nt DNA in 5′ and 16-nt DNA in 3′, avoiding the Cas9 binding region) mediates efficient genome editing. 3′ all DNA (22DNA) abolished genome editing. n = 3 biologically independent samples. Error bars show mean ± s.d.