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Here, we describe a genome-editing tool based on the temporal and conditional stabilization of clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) under the small molecule, Shield-1. The method can be used for cultured cells and animal models.
The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.
CRISPR-Cas9 which stands for "clustered regularly interspaced short palindromic repeats-associated protein 9" was first discovered as part of studies on bacterial adaptive immunity1,2. Today, CRISPR/Cas9 has become the most recognized tool for programmable gene editing and different iterations of the system have been developed to allow transcriptional and epigenetic modulations3. This technology enables the highly precise genetic manipulation of almost any sequence of DNA4.
The essential components of any CRISPR gene e....
1.The DD-Cas9 vector
To enable the conditional expression of Cas9, we developed a dual lentiviral vector construct consisting of a U6-driven promoter to constitutively express sgRNA, and an EF-1α core promoter to drive the expression of the DD-Cas9 fusion protein (Figure 1A)19. As a paradigm to illustrate the robustness and efficiency of the system, we transduced the lung carcinomatous A549 cell line with the lentiviral construct. The levels of Cas9 i.......
The CRISPR/Cas9 technology has revolutionized the capability of functionally interrogate genomes2. However, the inactivation of genes often results in cell lethality, functional deficits, and developmental defects, limiting the utility of such approaches for studying gene functions7. Additionally, constitutive expression of Cas9 may result in toxicity and the generation of off-target effects6. Different approaches have been developed to temporally co.......
We thank previous members of our laboratory and scientist Serif Senturk for previous work. We thank Danilo Segovia for critically reading this manuscript. This study was possible and supported by Swim Across America and the National Cancer Institute Cancer Target Discovery and Development Center program.
....Name | Company | Catalog Number | Comments |
100mM DTT | Thermosfisher | ||
10X FastDigest buffer | Thermosfisher | B64 | |
10X T4 Ligation Buffer | NEB | M0202S | |
colorimetric BCA kit | Pierce | 23225 | |
DMEM, high glucose, glutaMax | Thermo Fisher | 10566024 | |
FastAP | Thermosfisher | EF0654 | |
FastDigest BsmBI | Thermosfisher | FD0454 | |
Flag [M2] mouse mAb | Sigma | F1804-50UG | |
Genomic DNA extraction kit | Macherey Nagel | 740952.1 | |
lipofectamine 2000 | Invitrogen | 11668019 | |
Phusion High-Fidelity DNA Polymerase | NEB | M0530S | |
oligonucleotides | Sigma Aldrich | ||
pMD2.G | Addgene | 12259 | |
polybrene | Sigma Aldrich | TR-1003-G | |
psPAX2 | Addgene | 12260 | |
QIAquick PCR & Gel Cleanup Kit | Qiagen | 28506 | |
secondary antibodies | LICOR | ||
Shield-1 | Cheminpharma | ||
Stbl3 competent bacterial cells | Thermofisher | C737303 | |
SURVEYOR Mutation Detection Kit | Transgenomic/IDT | ||
T4 PNK | NEB | M0201S | |
Taq DNA Polymerase | NEB | M0273S | |
α-tubulin [DM1A] mouse mAb | Millipore | CP06-100UG |
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