Efficiënte generatie van iPSC Neural Lineage Specifieke Knockin Reporters Met de CRISPR / Cas9 en Cas9 Double Nickase System
Summary
Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.
Abstract
Gene targeting is een kritische benadering voor het karakteriseren van gen functies in het moderne biomedisch onderzoek. Echter, de efficiëntie van gen targeting in humane cellen laag is, waardoor de vorming van humane cellijnen voorkomen met een gewenste snelheid. De afgelopen twee jaar hebben een snelle progressie op het verbeteren van de efficiëntie van genetische manipulatie door genoom editing tools getuige zoals het CRISPR (regelmatig Gegroepeerd interspaced Short Palindromic Repeats) / Cas (-CRISPR geassocieerd) systeem. Dit manuscript beschrijft een protocol voor het genereren van lineage specifieke mens geïnduceerde pluripotente stamcellen (iPSC) reporters met CRISPR / Cas-systeem bijgestaan homologe recombinatie. Procedures voor het verkrijgen van de noodzakelijke componenten voor het maken van neurale lineage reporter lijnen met behulp van de CRISPR / Cas-systeem, gericht op de bouw van targeting vectoren en enkele gids RNA's, worden beschreven. Dit protocol kan worden uitgebreid tot platform vestiging en mutatie correctie hiPSCs.
Introduction
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, together with other genome editing tools, has revolutionized the human genome manipulation in recent years1-7. Major components in the CRISPR/Cas9 system are single guide RNA (sgRNA) and Cas9. Cas9 is an RNA-guided type II DNA endonuclease. It has two nuclease domains, HNH and RuvC, which are responsible for making DNA double-stranded breaks (DSBs) at a specific genomic region next to a protospacer adjacent motif (PAM)7-10.An sgRNA has a combined function of that of crRNA and tracrRNA, two adaptive immunity RNA molecules identified in bacteria (such as Streptococcus pyogenes) and archaea to fight against DNA invasion of exogenous sources9-11. When appropriately designed and co-introduced to mammalian cells, the sgRNA recognizes the PAM sequence, base-pairs with the complementary strand of target DNA, and guides and transactivates Cas9 to cleave at both DNA strands immediately upstream of PAM7. Subsequently, homologous directed recombination (HDR, in the presence of a targeting vector) or non-homologous end joining (NHEJ) will occur to repair the DSBs. Transgenes such as cDNAs, reporter cassettes, or antibiotic resistant fragments can be integrated, and transgenic or knockin lines made.
Although the CRISPR/Cas9 system is highly efficient and relatively specific, undesired off-target activities have been reported12-15. To minimize off-target events, Cas9 nickases (Cas9n) have also been engineered9,10,14,16,17. Cas9n (Cas9 D10A or Cas9 H840A) is Cas9 enzyme with mutations at one of the two nuclease domains, which only elicits a nick at one strand of DNA. To achieve cleavage at both strands, two sgRNAs are designed to guide a pair of nickases, which cuts separately at nearby loci of the two different DNA strands. The “double nicking” strategy requires a more stringent design than the conventional Cas9 platform, and offers both high efficiency and high specificity for gene editing experiments.
This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporter using CRISPR/Cas system assisted homologous recombination. Steps for obtaining the necessary components for making neural lineage reporter lines using the CRISPR/Cas system mediated genome editing are described, with a focus on construction of targeting vectors and sgRNAs. This protocol has been successfully repeated in multiple hiPSC lines including those derived from healthy individuals and from patients with CNS diseases. Genes targeted in our laboratory include OLIG2, HB9 (MNX1), NEUROG2, SOX1, ALDH1L1 and SOD1. Targeting efficiency from CRISPR/Cas system mediated homologous recombination in hiPSCs ranges from 20-40%, which is consistent with previous reports1,18,19. Compared to a typical efficiency of 1-2% in conventional gene targeting experiments, the targeting efficiency is significantly improved.
Protocol
Representative Results
Discussion
Gene targeting is an essential tool in characterizing gene functions. However, the relatively low efficiency demands labor-intensive and time-consuming work. Recently development on genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system has greatly improved the targeting efficiency. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporter using CRISPR/Cas system assisted homologous recombination. Steps …
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Department of Neurosurgery, Memorial Hermann Foundation Staman Ogilvie Fund, the Bentsen Stroke Center at the University of Texas Health Science Center at Houston, and Mission Connect TIRR Foundation.
Materials
| Name of Material/Equipment | Vendor | Catalog no. |
| 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 8×10 | 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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