Summary

HiPSC Sinir Lineage Özgül Knockin Muhabirleri Verimli Nesil kullanma CRISPR / Cas9 ve Cas9 Çift Nickase Sistemi

Published: May 28, 2015
doi:

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

Gen hedefleme modern biyomedikal araştırmalarda gen fonksiyonlarını karakterize için kritik bir yaklaşımdır. Bununla birlikte, insan hücrelerinde gen hedeflemesi etkinliği arzu edilen bir oranda insan hücre hatlarının üretilmesini engelleyen, düşük olmuştur. Geçtiğimiz iki yıl gibi CRISPR (interspaced Kısa palindromic Tekrarlar Düzenli Kümelenmiş) / Cas (CRISPR ilişkili) sistem olarak genom düzenleme araçları ile genetik manipülasyon verimliliğini artırmak üzerine hızlı bir ilerleme tanık olduk. Bu el yazması homolog rekombinasyon destekli CRISPR / Cas sistemini kullanarak soyu belirli insan uyarılmış pluripotent kök hücre (hiPSC) gazetecilere üretmek için bir protokol tanımlamaktadır. CRISPR / Cas sistemi kullanılarak nöral nesilli raportör çizgileri yapmak için gerekli bileşenleri elde hedefleme vektörleri ve tek bir kılavuz RNA'lar yapımı odaklanan prosedürleri tarif edilmiştir. Bu protokol hiPSCs platform kurulması ve mutasyon düzeltme uzatılabilir.

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

1. Design and Vector Construction for Targeting Vectors Once the lineage specific marker is determined, locate the genomic sequence of the gene and design the homology arms accordingly. The length of 5’ homology arm is ~1 kb and of 3’ homology arm is ~1.5 kb (Figure 1). Tag the reporter cassette sequentially downstream of the genomic sequence right before the stop codon, by subcloning, so that the endogenous expression is not altered or disrupted. Link the reporter, or d…

Representative Results

Targeting vectors with all necessary components including homology arms, reporter cassette, positive and negative selection cassettes are constructed (Figure 1). Based on the endogenous genomic locus, multiple (2 to 3) sgRNAs (if using the Cas9 system) or sgRNA pairs (if using the Cas9n double nickase strategy) are designed and constructed into human-gRNA-expression vector MLM3636 or pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A) backbones (Figure 2). Before transfecting hiPSCs, the sgRNAs are…

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 …

Disclosures

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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Cite This Article
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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