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Developmental Biology

Génération efficace de hiPSC neurones Lineage Reporters Knockin spécifique en utilisant l'CRISPR / Cas9 et Cas9 Système Double Nickase

Published: May 28, 2015 doi: 10.3791/52539
*1,2, *1,2, 1,2,5, 1,2,6, 1,2,4,7, 1,2,3
* These authors contributed equally

Materials

Name Company Catalog Number Comments
pStart-K Addgene 20346
pWS-TK6  Addgene 20350
pKD3  Addgene 45604
pKD46 The Coli Genetic Stock Center, CGSC 7634
PGK-neo-bpA sequence  Addgene 13442
Human BAC clones of target genes  https://bacpac.chori.org
JDS246 (Cas9-003), Mammalian codon-optimized streptococcus pyogenes Cas9-3X Flag Addgene 43861
MLM3636, Human-gRNA-ExpressionVector with U6 promoter  Addgene 43860
pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) Addgene 42335
sgRNA design, ZiFit http://zifit.partners.org/ZiFiT/
Off target prediction tool CasOT http://eendb.zfgenetics.org/casot/download.php
Perl http://www.perl.org/get.html
NCBI http://www.ncbi.nlm.nih.gov/
BLAT https://genome.ucsc.edu/cgi-bin/hgBlat?command=start
sgRNA Primer synthesis Sigma
One shot Top10 Electrocomp E. Coli Competent cells Life Technologies C4040-50
AccuPrime Pfx SupperMix Life Technologies 12344-040
Restriction enzymes NEB and Life Technologies
Zymoclean Gel DNA Recovery Kit Zymo Researech D4007
DNA quick extraction buffer  Epicentre QE0905T
Herculase II Fusion DNA Polymerases Agilent Technologies 600675
Digestion buffer 2 New England Biolab
6X DNA Loading Dye Thermo Scientific R0611
TeSR-E8 Kit for hESC/hiPSC Maintenance Stem Cell Technologies 05940
UltraPure Agarose  Life Technologies 16500-100
50xTAE Life Technologies B49
Essential 8 medium  Life Technologies A1517001
Dulbecco’s Phosphate Buffered Saline without Calcium and Magnesium  Life Technologies A12856-01
D-MEM/F12 with Glutamax  Life Technologies 10565018
D-MEM with Glutamax  Life Technologies 10566040
Fetal Bovine Serum-ES cell qualified  Life Technologies 10439
Knockout serum replacement  Life Technologies 10828010
2-mercaptoethanol 1000X  Life Technologies 21985023
Non Essential Amino Acid  Life Technologies 11140050
Stempro Accutase  Life Technologies A1110501
Dispase  Life Technologies 17105-041
0.25% Trypsin- EDTA solution  Life Technologies 25200-056
Geltrex  Life Technologies    12760-013
ROCK inhibitor Y-27632 Millipore    SCM075
SMC4 reagent  BD 354357
Neomycin resistant MEF  Millipore PMEF-NL
Hygromycin resistant MEF  Millipore PMEF-HL
G418 (Geneticin) LifeTechnologies 11811
Hygromycin B  Life Technologies 10687010
FIAU (Fialuridine, 1-2-Deoxy-2-fluoro-ß-D-arabinofuranosyl-5-iodouracil  Moravek Biochemicals and Radiochemicals    M251
Electroporator: Gene Pulser Xcell  Bio-Rad
0.4 cm electroporation cuvette  Bio-Rad 165-2088
DIG-High prime DNA labeling and detection starter kit II  Roche 11585614910
Hybridization denature solution  VWR 82021-478
PCR DIG probe synthesis kit  Roche 11636090910
DIG wash set  Roche 11585762001
Anti-Digoxigenin (DIG-AP) Roche 11093274910
CSPD chemiluminescence system  Roche 11755633001
DIG wash and block buffer set  Roche 11585762001
50X TAE buffer  Life Technologies 24710030
Blotting buffer (25 mM Tris pH 7.4,  0.15 M NaCl,  0.1% Tween20)
Hoefer Ultraviolet Crosslinker  Fisher Scientific 03-500-308
Spermidine  Fisher AC13274-0010
Tris HCl 2M (pH 7.5) VWR    200064-506
Denville Scientific blue bio film 8x10  Fisher nc9550782
DNA molecular weight marker II ( DIG-labeled ) Roche 11218590910
Amersham Blotting membrane Hybond-N+  Roche 95038-400
Pyrex glass drying tray  Fisher 15-242A
Kimberly-Clark C-fold paper towels  Fisher 06-666-32B
Whatman 3MM paper (26X41) Fisher    05-713-336
Hybridization bag  Roche 11666649001
Hybridization tubes  Fisher 13-247-300
Hybridization oven rotisserie Shake 'n' Stack  Fisher HBMSOV14110

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References

  1. Wang, H., et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 153, 910-918 (2013).
  2. Mali, P., et al. RNA-guided human genome engineering via Cas9. Science. 339, 823-826 (2013).
  3. Hwang, W. Y., et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature. 31, 227-229 (2013).
  4. Friedland, A. E., et al. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods. , (2013).
  5. DiCarlo, J. E., et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336-4343 (2013).
  6. Cong, L., et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339, 819-823 (2013).
  7. Anders, C., Niewoehner, O., Duerst, A., Jinek, M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 513, 569-573 (2014).
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  9. Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America. 109, E2579-E2586 (2012).
  10. Jinek, M., et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337, 816-821 (2012).
  11. Jinek, M., et al. RNA-programmed genome editing in human cells. eLife. 2, e00471 (2013).
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  13. Hsu, P. D., et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology. 31, 827-832 (2013).
  14. Mali, P., et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 31, 833-838 (2013).
  15. Pattanayak, V., et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology. 31, 839-843 (2013).
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  17. Ran, F. A., et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 154, 1380-1389 (2013).
  18. Yang, H., Wang, H., Jaenisch, R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nature Protocols. 9, 1956-1968 (2014).
  19. Yang, H., et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 154, 1370-1379 (2013).
  20. Szymczak, A. L., et al. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nature Biotechnology. 22, 589-594 (2004).
  21. Carey, B. W., et al. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proceedings of the National Academy of Sciences of the United States of America. 106, 157-162 (2009).
  22. Yagi, T., et al. A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene. Analytical Biochemistry. 214, 77-86 (1993).
  23. Wu, S., Ying, G., Wu, Q., Capecchi, M. R. A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond. Nature Protocols. 3, 1056-1076 (2008).
  24. Xue, H., et al. A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells. Stem Cells. 27, 1836-1846 (2009).
  25. Liu, Y., Jiang, P., Deng, W. OLIG gene targeting in human pluripotent stem cells for motor neuron and oligodendrocyte differentiation. Nature Protocols. 6, 640-655 (2011).
Génération efficace de hiPSC neurones Lineage Reporters Knockin spécifique en utilisant l'CRISPR / Cas9 et Cas9 Système Double Nickase
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Cite this Article

Li, S., Xue, H., Long, B., Sun, L.,More

Li, S., Xue, H., Long, B., Sun, L., Truong, T., Liu, Y. Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System. J. Vis. Exp. (99), e52539, doi:10.3791/52539 (2015).

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