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Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system

Sakuma, Tetsushi, Nishikawa, Ayami, Kume, Satoshi, Chayama, Kazuaki, Yamamoto, Takashi·2014·Scientific Reports
DOI 10.1038/srep05400PMC PMC4066266
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Figure 1 Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome engineering. Oligonucleotides corresponding to each target sequence are annealed and inserted into BpiI-digested pX330A or pX330S vectors (STEP 1). The constructed vectors harboring single gRNA expression cassettes are then assembled into an all-in-one vector harboring multiple gRNA cassettes using the Golden Gate assembly method (STEP 2). Amp, ampicillin; Spec, spectinomycin; U6, human U6 promoter; CBh, chicken beta-actin short promoter.
Figure 1 Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome engineering. Oligonucleotides corresponding to each target sequence are annealed and inserted into BpiI-digested pX330A or pX330S vectors (STEP 1). The constructed vectors harboring single gRNA expression cassettes are then assembled into an all-in-one vector harboring multiple gRNA cassettes using the Golden Gate assembly method (STEP 2). Amp, ampicillin; Spec, spectinomycin; U6, human U6 promoter; CBh, chicken beta-actin short promoter.CC BY-NC-SA · PubMed Central
Figure 2 Multiplex genome editing with Cas9 nuclease and seven gRNAs. (A) Schematic illustration of the all-in-one vector expressing seven gRNAs targeting seven different genomic loci and Cas9 nuclease. The blue and green bent arrows indicate the U6 and CBh promoters, respectively. (B) Genomic cleavage analysis of the seven genomic loci targeted with the all-in-one CRISPR/Cas9-nuclease vector. The products from untransfected control cells (C) and cells transfected with CRISPR/Cas9-nuclease vectors targeting seven (7) and single (1) loci were analyzed by agarose gel electrophoresis. The percentage of non-homologous end-joining (% NHEJ) was estimated using ImageJ software as previously described30.
Figure 2 Multiplex genome editing with Cas9 nuclease and seven gRNAs. (A) Schematic illustration of the all-in-one vector expressing seven gRNAs targeting seven different genomic loci and Cas9 nuclease. The blue and green bent arrows indicate the U6 and CBh promoters, respectively. (B) Genomic cleavage analysis of the seven genomic loci targeted with the all-in-one CRISPR/Cas9-nuclease vector. The products from untransfected control cells (C) and cells transfected with CRISPR/Cas9-nuclease vectors targeting seven (7) and single (1) loci were analyzed by agarose gel electrophoresis. The percentage of non-homologous end-joining (% NHEJ) was estimated using ImageJ software as previously described30.CC BY-NC-SA · PubMed Central
Figure 3 Multiplex genome editing with Cas9 nickase and six gRNAs. (A) Schematic illustration of the all-in-one vector expressing six gRNAs targeting three different genomic loci and Cas9 nuclease. The black box indicates exon 14 of the human APC gene. The numbers from 2118 to 10804 represent the base positions in the APC gene transcript (NCBI reference sequence: NM_001127511.2). (B) The sizes of PCR products and cut images in each locus. (C) Genomic cleavage analysis of the three genomic loci targeted with the all-in-one CRISPR/Cas9-nickase vector. The products from untransfected control cells (C) and cells transfected with the CRISPR/Cas9-nickase vector expressing six gRNAs (6) were analyzed by agarose gel electrophoresis. The arrowheads indicate the approximate positions of the cleaved fragments. % NHEJ was estimated using ImageJ software as previously described30. W, Wide-Range DNA Ladder (100–2,000 bp) (Takara Bio, Shiga, Japan).
Figure 3 Multiplex genome editing with Cas9 nickase and six gRNAs. (A) Schematic illustration of the all-in-one vector expressing six gRNAs targeting three different genomic loci and Cas9 nuclease. The black box indicates exon 14 of the human APC gene. The numbers from 2118 to 10804 represent the base positions in the APC gene transcript (NCBI reference sequence: NM_001127511.2). (B) The sizes of PCR products and cut images in each locus. (C) Genomic cleavage analysis of the three genomic loci targeted with the all-in-one CRISPR/Cas9-nickase vector. The products from untransfected control cells (C) and cells transfected with the CRISPR/Cas9-nickase vector expressing six gRNAs (6) were analyzed by agarose gel electrophoresis. The arrowheads indicate the approximate positions of the cleaved fragments. % NHEJ was estimated using ImageJ software as previously described30. W, Wide-Range DNA Ladder (100–2,000 bp) (Takara Bio, Shiga, Japan).CC BY-NC-SA · PubMed Central
Figure 4 Large deletions mediated by all-in-one CRISPR/Cas9 vectors. (A) Schematic illustration of large deletion at the HPRT1 locus. The black boxes indicate exons. The blue lines and letters indicate PCR products. The zigzag lines indicate the HPRT1_A and HPRT1_B target sites. (B) Schematic illustration of large deletion at the APC locus. The black boxes indicate exons. The blue lines and letters indicate PCR products. The triangles indicate the off-5, off-8 and off-4 target sites. (C) Genomic PCR analysis of the HPRT1 and APC loci. The products from untransfected control cells (C) and cells transfected with the CRISPR/Cas9-nuclease vector expressing seven gRNAs (7) or the CRISPR/Cas9-nickase vector expressing six gRNAs (6) were analyzed by agarose gel electrophoresis. Red and blue asterisks indicate PCR products from un-deleted alleles, whereas yellow and green asterisks indicate PCR products from chromosomally deleted alleles. λ, λHindIII marker. W, Wide-Range DNA Ladder (100–2,000 bp) (Takara Bio). (D) Sequences of the PCR products from deleted alleles at the HPRT1 locus. The HPRT1_A and HPRT1_B target sites are indicated by red letters. PAM sites are indicated by black boxes. Deletions are indicated by dashes. (E) Sequences of the PCR products from deleted alleles at the APC locus. The off-5_L and off-4_R target sites are indicated by red letters. PAM sites are indicated by black boxes. Deletions are indicated by dashes.
Figure 4 Large deletions mediated by all-in-one CRISPR/Cas9 vectors. (A) Schematic illustration of large deletion at the HPRT1 locus. The black boxes indicate exons. The blue lines and letters indicate PCR products. The zigzag lines indicate the HPRT1_A and HPRT1_B target sites. (B) Schematic illustration of large deletion at the APC locus. The black boxes indicate exons. The blue lines and letters indicate PCR products. The triangles indicate the off-5, off-8 and off-4 target sites. (C) Genomic PCR analysis of the HPRT1 and APC loci. The products from untransfected control cells (C) and cells transfected with the CRISPR/Cas9-nuclease vector expressing seven gRNAs (7) or the CRISPR/Cas9-nickase vector expressing six gRNAs (6) were analyzed by agarose gel electrophoresis. Red and blue asterisks indicate PCR products from un-deleted alleles, whereas yellow and green asterisks indicate PCR products from chromosomally deleted alleles. λ, λHindIII marker. W, Wide-Range DNA Ladder (100–2,000 bp) (Takara Bio). (D) Sequences of the PCR products from deleted alleles at the HPRT1 locus. The HPRT1_A and HPRT1_B target sites are indicated by red letters. PAM sites are indicated by black boxes. Deletions are indicated by dashes. (E) Sequences of the PCR products from deleted alleles at the APC locus. The off-5_L and off-4_R target sites are indicated by red letters. PAM sites are indicated by black boxes. Deletions are indicated by dashes.CC BY-NC-SA · PubMed Central

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Citations

Cites

Research works printed but not linked: 18

  1. Sakuma, T. & Woltjen, K. Nuclease-mediated genome editing: At the front-line of functional genomics technology. Dev. Growth Differ. 56, 2–13 (2014). · DOI
  2. Carroll, D. Genome engineering with targetable nucleases. Annu. Rev. Biochem. 83, 409–439 (2014). · DOI · Paper page
  3. Guschin, D. Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010). · DOI
  4. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013). · DOI · Paper page
  5. Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014). · DOI · Paper page
  6. Mashiko, D. et al. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci. Rep. 3, 3355 (2013). · DOI
  7. Mashiko, D. et al. Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes. Dev. Growth Differ. 56, 122–129 (2014). · DOI
  8. Fujii, W., Onuma, A., Sugiura, K. & Naito, K. Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system. Biochem. Biophys. Res. Commun. 445, 791–794 (2014). · DOI
  9. Zhou, J. et al. Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting. FEBS J. 281, 1717–1725 (2014). · DOI
  10. Shen, B. et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods 11, 399–402 (2014). · DOI · Paper page
  11. Malina, A. et al. Repurposing CRISPR/Cas9 for in situ functional assays. Genes Dev. 27, 2602–2614 (2013). · DOI
  12. Cheng, A. W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013). · DOI
  13. Rusk, N. CRISPRs and epigenome editing. Nat. Methods 11, 28 (2014). · DOI
  14. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013). · DOI
  15. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011). · DOI
  16. Sakuma, T. et al. Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes Cells 18, 315–326 (2013). · DOI
  17. Sakuma, T. et al. Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Sci. Rep. 3, 3379 (2013). · DOI
  18. Hansen, K. et al. Genome editing with CompoZr custom zinc finger nucleases (ZFNs). J. Vis. Exp. e3304 (2012). · DOI

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Metadata

Authors
Sakuma, T., Nishikawa, A., Kume, S., Chayama, K., & Yamamoto, T.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2014-06-23
Volume
4
Issue
1
ISSN
2045-2322
DOI
10.1038/srep05400
PMC
PMC4066266
Cited by
352
References
12
Language
en
Licence

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