DNA targeting specificity of RNA-guided Cas9 nucleases - PubMed (original) (raw)
doi: 10.1038/nbt.2647. Epub 2013 Jul 21.
David A Scott, Joshua A Weinstein, F Ann Ran, Silvana Konermann, Vineeta Agarwala, Yinqing Li, Eli J Fine, Xuebing Wu, Ophir Shalem, Thomas J Cradick, Luciano A Marraffini, Gang Bao, Feng Zhang
Affiliations
- PMID: 23873081
- PMCID: PMC3969858
- DOI: 10.1038/nbt.2647
DNA targeting specificity of RNA-guided Cas9 nucleases
Patrick D Hsu et al. Nat Biotechnol. 2013 Sep.
Abstract
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
Figures
Figure 1
Optimization of guide RNA architecture for SpCas9-mediated mammalian genome editing. (a) Schematic of bicistronic expression vector (PX330) for U6 promoter-driven sgRNA and CBh promoter-driven human codon-optimized S. pyogenes Cas9 (hSpCas9) used for all subsequent experiments. The sgRNA consists of a 20-nt guide sequence (blue) and scaffold (red), truncated at various positions as indicated. (b) SURVEYOR assay for SpCas9-mediated indels at the human EMX1 and PVALB loci. Arrowheads indicate the expected SURVEYOR fragments (n = 3). (c) Northern blot analysis for the four sgRNA truncation architectures, with U1 as loading control. (d) Both wild-type (WT) or nickase mutant (D10A) of SpCas9 promoted insertion of a HindIII site into the human EMX1 gene. Single-stranded oligonucleotides, oriented in either the sense or antisense direction relative to genome sequence, were used as homologous recombination templates (Supplementary Fig. 3). (e) Schematic of the human SERPINB5 locus. sgRNAs and PAMs are indicated by colored bars above sequence; methylcytosine (Me) are highlighted (pink) and numbered relative to the transcriptional start site (TSS, +1). (f) Methylation status of SERPINB5 assayed by bisulfite sequencing of 16 clones. Filled circles, methylated CpG; open circles, unmethylated CpG. (g) Modification efficiency by three sgRNAs targeting the methylated region of SERPINB5, assayed by deep sequencing (n = 2). Error bars indicate Wilson intervals (Online Methods).
Figure 2
Single-nucleotide specificity of SpCas9. (a) Schematic of the experimental design. sgRNAs carrying all possible single base-pair mismatches (blue Ns) throughout the guide sequence were tested for each EMX1 target site (target site 1 shown as example). (b) Heatmap representation of relative SpCas9 cleavage efficiency by 57 single-mutated and 1 nonmutated sgRNA each for four EMX1 target sites (aggregated from Supplementary Table 5). For each EMX1 target, the identities of single base-pair substitutions are indicated on the left; original guide sequence is shown above and highlighted in the heatmap (gray squares). Modification efficiencies (increasing from white to dark blue) are normalized to the original guide sequence. Sequence logo representation of the same data can be found in Supplementary Figure 7. (c) Heatmap for relative SpCas9 cleavage efficiency for each possible RNA:DNA base pair, compiled from aggregate data from single-mismatch guide RNAs for 15 EMX1 targets (Supplementary Fig. 8). Mean cleavage levels were calculated for the 10 PAM-proximal bases (right bar) and across all substitutions at each position (bottom bar); positions in gray were not covered by the 469 single-mutated and 15 unmutated sgRNAs tested (Supplementary Table 5). (d) SpCas9-mediated indel frequencies at targets with all possible PAM sequences, determined using the SURVEYOR nuclease assay. Two target sites from the EMX1 locus were tested for each PAM (Supplementary Table 4). (e) Histogram of distances between 5′-NRG PAM occurrences within the human genome. Putative targets were identified using both strands of human chromosomal sequences (GRCh37/hg19).
Figure 3
Multiple mismatch specificity of SpCas9. (a–c) SpCas9 cleavage efficiency with guide RNAs containing consecutive mismatches of 2, 3 or 5 bases (a), or multiple mismatches separated by different numbers of unmutated bases for EMX1 targets 1, 2, 3 and 6 (b, c). Rows represent each mutated guide RNA; nucleotide substitutions are shown in white cells; gray cells denote unmutated bases. All indel frequencies are absolute and analyzed by deep sequencing from two biological replicas. Error bars indicate Wilson intervals (Online Methods).
Figure 4
SpCas9-mediated indel frequencies at predicted genomic off-target loci. (a, b) Cleavage levels at putative genomic off-target loci containing two or three individual mismatches (white cells) for EMX1 target 1 and target 3 are analyzed by deep sequencing. List of off-target sites are ordered by median position of mutations. Putative off-target sites with additional mutations did not have detectable indels (Supplementary Table 8). The Cas9 dosage was 3 × 10−10 nmol/cell, with equimolar sgRNA delivery. Error bars indicate Wilson intervals (Online Methods). (c, d) Indel frequencies for EMX1 targets 1 and 3 and selected off-target loci (OT) as a function of SpCas9 and sgRNA dosage, (n = 2, Wilson intervals). 400 ng to 10 ng of Cas9-sgRNA plasmid corresponds to 7.1 × 10−10 to 1.8 × 10−11 nmol/cell. Cleavage specificity is measured as a ratio of on- to off-target cleavage.
Comment in
- Staying on target with CRISPR-Cas.
Carroll D. Carroll D. Nat Biotechnol. 2013 Sep;31(9):807-9. doi: 10.1038/nbt.2684. Nat Biotechnol. 2013. PMID: 24022156 No abstract available.
Similar articles
- High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. Kleinstiver BP, et al. Nature. 2016 Jan 28;529(7587):490-5. doi: 10.1038/nature16526. Epub 2016 Jan 6. Nature. 2016. PMID: 26735016 Free PMC article. - CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.
Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G. Lin Y, et al. Nucleic Acids Res. 2014 Jun;42(11):7473-85. doi: 10.1093/nar/gku402. Epub 2014 May 16. Nucleic Acids Res. 2014. PMID: 24838573 Free PMC article. - High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.
Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. Pattanayak V, et al. Nat Biotechnol. 2013 Sep;31(9):839-43. doi: 10.1038/nbt.2673. Epub 2013 Aug 11. Nat Biotechnol. 2013. PMID: 23934178 Free PMC article. - Origins of Programmable Nucleases for Genome Engineering.
Chandrasegaran S, Carroll D. Chandrasegaran S, et al. J Mol Biol. 2016 Feb 27;428(5 Pt B):963-89. doi: 10.1016/j.jmb.2015.10.014. Epub 2015 Oct 23. J Mol Biol. 2016. PMID: 26506267 Free PMC article. Review. - Cas9 as a versatile tool for engineering biology.
Mali P, Esvelt KM, Church GM. Mali P, et al. Nat Methods. 2013 Oct;10(10):957-63. doi: 10.1038/nmeth.2649. Nat Methods. 2013. PMID: 24076990 Free PMC article. Review.
Cited by
- Cleavage of DNA Substrate Containing Nucleotide Mismatch in the Complementary Region to sgRNA by Cas9 Endonuclease: Thermodynamic and Structural Features.
Baranova SV, Zhdanova PV, Koveshnikova AD, Pestryakov PE, Vokhtantsev IP, Chernonosov AA, Koval VV. Baranova SV, et al. Int J Mol Sci. 2024 Oct 9;25(19):10862. doi: 10.3390/ijms251910862. Int J Mol Sci. 2024. PMID: 39409191 Free PMC article. - CRISPR/Cas9 gene editing in induced pluripotent stem cells to investigate the feline hypertrophic cardiomyopathy causing MYBPC3/R820W mutation.
Dutton LC, Dudhia J, Guest DJ, Connolly DJ. Dutton LC, et al. PLoS One. 2024 Oct 10;19(10):e0311761. doi: 10.1371/journal.pone.0311761. eCollection 2024. PLoS One. 2024. PMID: 39388496 Free PMC article. - Making gene editing accessible in resource limited environments: recommendations to guide a first-time user.
Goolab S, Scholefield J. Goolab S, et al. Front Genome Ed. 2024 Sep 25;6:1464531. doi: 10.3389/fgeed.2024.1464531. eCollection 2024. Front Genome Ed. 2024. PMID: 39386178 Free PMC article. Review. - Gene editing in livestock: innovations and applications.
Rodriguez-Villamil P, Beaton BP, Krisher RL. Rodriguez-Villamil P, et al. Anim Reprod. 2024 Sep 23;21(3):e20240054. doi: 10.1590/1984-3143-AR2024-0054. eCollection 2024. Anim Reprod. 2024. PMID: 39372257 Free PMC article. - Candidate gene analysis of rice grain shape based on genome-wide association study.
Xin W, Chen N, Wang J, Liu Y, Sun Y, Han B, Wang X, Liu Z, Liu H, Zheng H, Yang L, Zou D, Wang J. Xin W, et al. Theor Appl Genet. 2024 Sep 29;137(10):241. doi: 10.1007/s00122-024-04724-8. Theor Appl Genet. 2024. PMID: 39342533
References
Publication types
MeSH terms
Substances
Grants and funding
- R01-CA133404/CA/NCI NIH HHS/United States
- HHMI/Howard Hughes Medical Institute/United States
- DP1-MH100706/DP/NCCDPHP CDC HHS/United States
- T32 GM007753/GM/NIGMS NIH HHS/United States
- R01-DK097768/DK/NIDDK NIH HHS/United States
- PN2EY018244/EY/NEI NIH HHS/United States
- R01 GM034277/GM/NIGMS NIH HHS/United States
- R01 DK097768/DK/NIDDK NIH HHS/United States
- R01-GM34277/GM/NIGMS NIH HHS/United States
- R01 CA133404/CA/NCI NIH HHS/United States
- DP1 MH100706/MH/NIMH NIH HHS/United States
LinkOut - more resources
Full Text Sources
Other Literature Sources