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In vitro Cleavage and Electrophoretic Mobility Shift Assays for Very Fast CRISPR

快速CRISPR的体外裂解和电泳迁移率位移测定

RZ Roger S. Zou
YL Yang Liu
TH Taekjip Ha

发布: 2021年09月05日第11卷第17期 DOI: 10.21769/BioProtoc.4138 浏览次数: 4832

评审: Pilar Villacampa AlcubierreWenyang Li

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Abstract

CRISPR-Cas9 has transformed biomedical research and medicine through convenient and targeted manipulation of DNA. Time- and spatially-resolved control over Cas9 activity through the recently developed very fast CRISPR (vfCRISPR) system have facilitated comprehensive studies of DNA damage and repair. Understanding the fundamental principles of Cas9 binding and cleavage behavior is essential before the widespread use of these systems and can be readily accomplished in vitro through both cleavage and electrophoretic mobility shift assays (EMSA). The protocol for in vitro cleavage consists of Cas9 with guide RNA (gRNA) ribonucleoprotein (RNP) formation, followed by incubation with target DNA. For EMSA, this reaction is directly loaded onto an agarose gel for visualization of the target DNA band that is shifted due to binding by the Cas9 RNP. To assay for cleavage, Proteinase K is added to degrade the RNP, allowing target DNA (cleaved and/or uncleaved) to migrate consistently with its molecular weight. Heating at 95°C rapidly inactivates the RNP on demand, allowing time-resolved measurements of Cas9 cleavage kinetics. This protocol facilitates the characterization of the light-activation mechanism of photocaged vfCRISPR gRNA.

Keywords: CRISPR-Cas9 (CRISPR-Cas9) search In vitro (活体外 ) search Cleavage (分裂) search Electrophoretic mobility shift assay (电泳迁移率实验) search EMSA (电泳迁移率实验) search Genome editing (基因组编辑) search

Background

Endonucleases, especially Cas9, from clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systems (Marraffini et al., 2015) have found widespread use as highly versatile tools in the biomedical sciences from genome editing, functional screening, to imaging (Adli et al., 2018). Recently, highly resolved spatiotemporal control over Cas9 activity via very fast CRISPR (vfCRISPR) has enabled detailed studies of the DNA damage response through highly synchronized activation of Cas9 in space and time (Liu et al., 2020). For all uses, characterization of these nucleases is essential and can be most readily accomplished in vitro (Singh et al., 2016). Electrophoretic mobility shift assays (EMSA) are powerful tools to characterize protein-DNA binding that can be directly applied to determine the binding affinity of Cas9 to nucleic acids (Hellman et al., 2007). In contrast, in vitro cleavage assays allow characterization of the Cas9 DNA cleavage mechanism. In this manuscript, we outline detailed protocols for both strategies to characterize the properties of the vfCRISPR light-inducible Cas9 system. We demonstrate how both EMSA and in vitro cleavage assays characterize the binding, speed, and efficiency of vfCRISPR, which paves the way for its use in biological systems.

Materials and Reagents

  1. 10 mg/ml SpCas9 (purified in-house from BL21 CodonPlus (DE3)-RIL cells [Agilent 230245], but can also be purchased from Integrated DNA Technologies) (Alt-R®, catalog number: 1081058, storage temperature: -80°C)

  2. tracrRNA (Integrated DNA Technologies) (Alt-R®, catalog number: 1072533, storage temperature: -80°C)

  3. Photocaged crRNA (cgRNA) from vfCRISPR targeting PPP1R2 (BioSynthesis, sequence: GACUUCCUCUAUGGUGGCGUGUUUUAGAGCUAUGCUGUUUUG; U corresponds to replacements of uracil with NPOM-dT photocaged nucleotides, storage temperature: -80°C)

  4. HEK293T cells (ATCC, catalog number: CRL-3216, storage temperature: liquid nitrogen vapor)

  5. Nuclease-Free Duplex Buffer (Integrated DNA Technologies, catalog number: 11-01-03-01, storage temperature: 4°C)

  6. PPP1R2 Fwd PCR primer (Integrated DNA Technologies, storage temperature: -20°C, sequence: 5’-GTTTCCGAGGCAGCAGTTG-3’)

  7. PPP1R2 Rev PCR primer (Integrated DNA Technologies, storage temperature: -20°C, sequence: 5’-GCATGATAAACGTCATCGCCC-3’)

  8. DNeasy Blood & Tissue Kit (Qiagen, DNeasy, catalog number: 69504)

  9. QIAquick PCR purification kit (Qiagen, QIAquick, catalog number: 28104)

  10. Q5® High-Fidelity 2X Master Mix (New England BioLabs, Q5®, catalog number: M0492, storage temperature: -20°C)

  11. NEBufferTM 3.1 (New England BioLabs, catalog number: B7203S, storage temperature: -20°C)

  12. Proteinase K, Molecular Biology Grade (New England BioLabs, catalog number: P8107S, storage temperature: -20°C)

  13. E-GelTM Agarose Gels with SYBRTM Safe DNA Gel Stain, 4% (Thermo Fisher, E-GelTM, catalog number: A45206, storage temperature: room temperature)

  14. 100 bp DNA ladder (New England BioLabs, catalog number: N3231S)

  15. HEPES (Sigma-Aldrich, catalog number: H3375)

  16. KCl (Sigma-Aldrich, catalog number: P3911)

  17. Glycerol (Sigma-Aldrich, catalog number: G5516)

  18. NaOH (Sigma-Aldrich, catalog number: 221465)

  19. Aluminum foil (Reynolds Wrap)

  20. Nuclease-Free Water (not DEPC-treated) (InvitrogenTM, catalog number: AM9932)

Equipment

  1. C1000 TouchTM Thermo Cycler (Bio-Rad, model number: 1851148), or any alternative thermocycler that can perform polymerase chain reaction (PCR)

  2. E-Gel® iBaseTM Power System (Thermo Fisher, E-GelTM, catalog number: G6465), or alternative gel electrophoresis system

  3. JAXMAN 365 nm LED flashlight (Amazon, JAXMAN, https://www.amazon.com/JAXMAN-Ultraviolet-365nm-Detector-Flashlight/dp/B06XW7S1CS/)

  4. TyphoonTM FLA 9000 (GE Healthcare), or any alternative gel imager that can image ethidium bromide or SYBR Gold agarose gels

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Zou, R. S., Liu, Y. and Ha, T. (2021). In vitro Cleavage and Electrophoretic Mobility Shift Assays for Very Fast CRISPR. Bio-protocol 11(17): e4138. DOI: 10.21769/BioProtoc.4138.
  2. Liu, Y., Zou, R. S., He, S., Nihongaki, Y., Li, X., Razavi, S., Wu, B. and Ha, T. (2020). Very fast CRISPR on demand. Science 368(6496): 1265-1269.

    3. ris ris Download Citation in RIS Format

分类

分子生物学 > DNA > DNA-蛋白质相互作用

生物科学 > 生物技术

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