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Peer-reviewed

Preparation of Cas9 Ribonucleoproteins for Genome Editing

用于基因组编辑的 Cas9 核糖核蛋白的制备

SL Sheng-Wei Lin
VN Viet Quoc Nguyen
SL Steven Lin

发布: 2022年05月20日第12卷第10期 DOI: 10.21769/BioProtoc.4420 浏览次数: 4738

评审: Wenzhi JiangRC WilsonAnonymous reviewer(s)

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Abstract

Genome editing by the delivery of pre-assembled Cas9 ribonucleoproteins (Cas9 RNP) is an increasingly popular approach for cell types that are difficult to manipulate genetically by the conventional plasmid and viral methods. Cas9 RNP editing is robust, precise, capable of multiplexing, and free of genetic materials. Its transient presence in cells limits residual editing activity. This protocol describes the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein by heterologous expression and purification from Escherichia coli, and the synthesis of CRISPR guide RNA by in vitro transcription and PAGE purification. SpCas9 is the first CRISPR Cas9 discovered (Jinek et al., 2012) and is also one of the most characterized Cas enzymes for genome editing applications. Using this formulation of Cas9 RNP, we have demonstrated highly efficient genome editing in primary human T and natural killer (NK) cells by electroporation, and in fungi and plants by polyethylene glycol-mediated transformation. Our protocol of Cas9 RNP preparation is consistent and straightforward to adopt for genome editing in other cell types and organisms.


Graphical abstract:



Keywords: CRISPR (CRISPR) search Cas9 RNP (Cas9 RNP) search sgRNA (sgRNA) search In vitro transcription (体外转录) search

Background

CRISPR-Cas9 genome editing is a revolutionary technology for research, biotechnology, agriculture, and medicine. Acting as molecular scissors, Cas9 nuclease induces a targeted DNA double-strand break at the site that is specified by the spacer sequence of guide RNA, creating a unique opportunity to modify genomic sequences through DNA repair pathways. The native form of Cas9 guide RNA consists of two RNA molecules: a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) (Jinek et al., 2012). The crRNA and tracrRNA were engineered into a single guide RNA (sgRNA) fusion molecule to facilitate in vitro and in vivo applications (Jinek et al., 2012). Genome editing by the delivery of pre-assembled Cas9 RNP is robust and precise, and is advantageous over the standard plasmid and viral approaches when transient editing activity is desired. Cas9 protein and guide RNA can be synthesized and validated in advance, and stably stored in the -80°C freezer for genome editing on demand. These attributes are ideal for the manufacture of genome-engineered immune cells that complies with good manufacturing practice standards for research and clinical applications.


We have established robust Cas9 RNP genome-editing platforms for primary human T and NK cells, using our highly pure, stable, and non-toxic SpCas9 protein and synthetic guide RNA (Schumann et al., 2015; Huang et al., 2020, 2021). Cas9 RNP can be introduced to the immune cells, by either electroporation in a Lonza 4D Nucleofector system or a ThermoFisher Neon Transfection system, to achieve high gene-knockout efficiency and cell viability. Multiple Cas9 RNP of different specificities can be delivered in a single reaction to simultaneously target multiple genomic loci. Furthermore, the co-electroporation of synthetic DNA repair templates enables site-specific gene knock-in by Cas9-mediated homology-directed repair. We have also demonstrated that the same Cas9 RNP formulation worked efficiently in fungi and plants by other delivery methods (Lai et al., 2017; Hsu et al., 2021). We believe that this protocol would be of great value to the scientific communities for a wide range of genome editing applications in mammalian cells as well as other organisms.


In this protocol, we describe the procedures for the heterologous expression and purification of recombinant SpCas9 and the enzymatic synthesis of sgRNA. Our protocol focuses on high purity and low toxicity of Cas9 RNP for applications in primary immune cells, which are more sensitive to endotoxin and exogenous RNA. Our protocol offers several improvements to the protocols by Savić et al. (2019) and Lingeman et al. (2017), including an extended His tag to increase binding to the nickel-affinity column, use of a mild detergent to reduce endotoxin contamination, improved ion exchange and gel-filtration chromatography, and sterilization of protein for cell culture use. Our protocol for the enzymatic synthesis of sgRNA includes denaturing PAGE purification to extract the full-length sgRNA and remove the truncated products. We also perform a phosphatase treatment to remove the immunogenic 5’ triphosphate moiety that is highly toxic to human immune cells (Wienert et al., 2018; Kim et al., 2018; Huang et al., 2021). We have shown that our guide RNA is of equally high quality to the commercial chemically synthesized guide RNA (Huang et al., 2021), but for only a fraction of the cost to prepare. An overview of Cas9 and sgRNA preparation is illustrated in Figure 1.



Figure 1. Overview of Cas9 and sgRNA synthesis and purification. (A) Cas9 is expressed in E. coli and purified from E. coli lysate by nickel-affinity, ion-exchange (heparin column), and gel-filtration chromatography. (B) sgRNA synthesis begins with the assembly of sgRNA DNA template by overlapping PCR using four DNA oligonucleotides: top (T), bottom (B), forward (F), and reverse (R). The DNA template is then transcribed in vitro by T7 RNA polymerase (T7 Pol) to generate sgRNA. After sgRNA purification by urea-PAGE, the immunogenic 5' triphosphate group is removed by treatment with calf intestinal phosphatase (CIP). The resulting Cas9 and sgRNA are mixed to form Cas9 ribonucleoproteins (Cas9 RNP) for genome editing applications.

Materials and Reagents

Materials

  1. Rainin pipette tips, 20-μL, 200-μL, and 1,000-μL (Mettler Toledo, catalog numbers: 30389291, 30389299 and 30389212)

  2. Serological pipettes, 5-mL, 10-mL, and 25-mL (ThermoFisher, catalog numbers: 170355N, 170356N and 170357N)

  3. 10-cm Petri dish (Fisher Scientific, catalog number: 08-757-105)

  4. 200-µL PCR tube (Gunster Biotech, catalog number: MB-P08A)

  5. Disposable plastic cuvette (BRAND, catalog number: 759115)

  6. 1.5-mL microcentrifuge tube (Axygen, catalog number: MCT-150-C)

  7. 15-mL conical tube (ThermoFisher, catalog number: 339650)

  8. 50-mL conical tube (ThermoFisher, catalog number: 339652)

  9. Nalgene Oak Ridge PPCO centrifuge tube, 42-mL (ThermoFisher, catalog number: 3119-0050)

  10. 1-L polypropylene centrifuge bottle (Beckman Coulter, catalog number: A98813)

  11. ColiRoller plating glass beads (Sigma-Aldrich, catalog number: 71013)

  12. Polyethersulfone Membrane Filters, 0.45-µm pore size, 47 mm diameter (Sartorius, catalog number: 15406-47-N)

  13. Millicup-FLEX filtration unit, 47 mm diameter (Millipore, catalog number: MCFLX4702)

  14. Ni-NTA Superflow resin (Qiagen, catalog number: 30450)

  15. Econo-column, 2.5 cm × 10 cm (Bio-Rad, catalog number: 7372512)

  16. Econo-column funnels (Bio-Rad, catalog number: 7310003)

  17. Clear 96-well microplate (PerkinElmer, catalog number: 6005640)

  18. Amicon Ultra-15, 100-kDa MWCO (Millipore, catalog number: UFC910096)

  19. 0.22-µm syringe filter, PES membrane, 33 mm diameter (Millipore, catalog number: SLGPR33RS)

  20. 0.45-µm syringe filter, PES membrane, 33 mm diameter (Millipore, catalog number: SLHPR33RS)

  21. HiTrap Heparin HP, 5-mL (Cytiva, catalog number: 17040703)

  22. HiLoad Superdex 200 16/600 (Cytiva, catalog number: 28-9893-35)

  23. Prefilter 6000 (GE Healthcare, catalog number: 18-4582-01)

  24. Ultra-free MC centrifugal filter, 0.2-µm (Millipore, catalog number: UFC30GV08)

  25. Mini-PROTEAN TGX precast gel (Bio-Rad, catalog number: 4561033)

  26. Kimwipes disposable wipers (Sigma-Aldrich, catalog number: Z188956)

  27. Large binder clip, 2” (Amazon, catalog number: B07V3VZLG1)

  28. Disposable syringes, 3-mL and 10-mL (Terumo, catalog numbers: SS-03S and SS-10S)

  29. 23G × 1 1/2" needle (Terumo, catalog number: NN-2338R)

  30. 22G × 2” metal hub needle (Hamilton, catalog number: 91022)

  31. Plastic food wrap (Amazon, catalog number: B00JPKW1RQ)

  32. Green-fluorescing F254 silica thin-layer chromatography plate (Sigma-Aldrich, catalog number: 02554)

  33. Disposable surgical blade (Feather Safety Razor, No. 10)


Reagents

  1. SpCas9 expression plasmid (Addgene, catalog number: 182032)

  2. Novagen E. coli Tuner (DE3) competent cells (Sigma-Aldrich, catalog number: 70623)

  3. Difco LB Broth, Miller (Luria-Bertani) (BD, catalog number: 244610)

  4. Terrific Broth (BD, catalog number: 243810)

  5. Agar, bacteriology (VWR, catalog number: J637)

  6. Glycerol (Fisher Scientific, catalog number: G/0650/17)

  7. Na2HPO4•7H2O (Sigma-Aldrich, catalog number: S9390)

  8. KH2PO4 (Sigma-Aldrich, catalog number: P0662)

  9. NaCl (Sigma-Aldrich, catalog number: S9888)

  10. NH4Cl (Sigma-Aldrich, catalog number: 213330)

  11. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (GoldBio, catalog number: I2481C100)

  12. Kanamycin monosulfate (GoldBio, catalog number: K-120-50)

  13. Difco Terrific broth (BD, catalog number: 243810)

  14. HEPES (Sigma-Aldrich, catalog number: 54457)

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

  16. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148)

  17. Imidazole (Sigma-Aldrich, catalog number: I2399)

  18. Triton X-100 (Sigma-Aldrich, catalog number: 93443)

  19. Lysozyme (Sigma-Aldrich, catalog number: L4919)

  20. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 10837091001)

  21. DNase I from bovine pancreas (Roche, catalog number: 11284932001)

  22. Liquid nitrogen (local distributor)

  23. NuPAGE LDS loading dye (ThermoFisher, catalog number: NP0007)

  24. Bradford assay reagent (VWR, catalog number: E530)

  25. TEV protease (Sigma-Aldrich, catalog number: T4455)

  26. 10× Tris-glycine-SDS buffer (Sigma-Aldrich, catalog number: T7777)

  27. ExcelBand protein marker (SMOBIO, catalog number: PM2500)

  28. InstantBlue Coomassie Protein Stain (Abcam, catalog number: 119211)

  29. DNA oligonucleotides for the synthesis of sgRNA template (IDT DNA)

  30. Qiagen EB buffer (Qiagen, catalog number: 19086)

  31. H2O, molecular biology grade (Fisher Scientific, catalog number: BP2819)

  32. KAPA HIFI PCR kit (Roche, catalog number: KK2103)

  33. 6× Gel Loading Dye Purple (NEB, catalog number: B7024S)

  34. Agarose I (VWR, catalog number: 0710)

  35. 10× TAE buffer (VWR, catalog number: K915)

  36. SYBR Safe DNA gel stain (ThermoFisher, catalog number: S33102)

  37. Diethylpyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: 40718)

  38. Tris (VWR, catalog number: 0826)

  39. MgCl2 hexahydrate (Sigma-Aldrich, catalog number: M2670)

  40. Spermidine (Sigma-Aldrich, catalog number: 85558)

  41. Polyethylene glycol (PEG) 2000 (Sigma-Aldrich, catalog number: 8.21037)

  42. Dithiothreitol (DTT) (Roche, catalog number: 10708984001)

  43. ATP (Sigma-Aldrich, catalog number: A2383)

  44. UTP (Sigma-Aldrich, catalog number: U6625)

  45. CTP (Sigma-Aldrich, catalog number: C1506)

  46. GTP (Sigma-Aldrich, catalog number: G8877)

  47. T7 RNA polymerase (Roche, catalog number: 10881767001)

  48. RQ1 RNase-free DNase I (Promega, catalog number: M6101)

  49. Formamide (Sigma-Aldrich, catalog number: F9037)

  50. Bromophenol blue (Sigma-Aldrich, catalog number: 114391)

  51. Xylene cyanol (Sigma-Aldrich, catalog number: X4126)

  52. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884)

  53. Urea (Sigma-Aldrich, catalog number: U5128)

  54. 5× TBE buffer (VWR, catalog number: J885)

  55. 40% polyacrylamide, 29:1 (ACROS, catalog number: 330215000)

  56. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678)

  57. N,N,N’,N’ Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281)

  58. Scienceware Aquet liquid detergent (Sigma-Aldrich, catalog number: Z273260)

  59. Sigmacote siliconizing reagent (Sigma-Aldrich, catalog number: SL2)

  60. 3 M sodium acetate solution, pH 5 (Sigma-Aldrich, catalog number: S7899)

  61. Glycogen (Abcam, catalog number: AB146544)

  62. Isopropanol (EMD Millipore, catalog number: 1.09634.2500)

  63. Ethanol (Katayama Chemical, catalog number: 9270)

  64. Quick CIP, calf intestinal alkaline phosphatase (NEB, catalog number: M0525)

  65. Phenol:chloroform mix, low pH (VWR, catalog number: 0966)

  66. Chloroform (Sigma-Aldrich, catalog number: C2432)

  67. ToxinSensor chromogenic lyophilized Amebocyte lysate (LAL) endotoxin assay kit (GeneScript, catalog number: L00350)

  68. HCl (Fisher Scientific, catalog number: A508-P500)

  69. LB+glycerol medium (see Recipes)

  70. Terrific Broth (see Recipes)

  71. 20× modified M9 buffer (see Recipes)

  72. 1 M IPTG (see Recipes)

  73. 50 mg/mL kanamycin (see Recipes)

  74. LB agar plate with kanamycin (see Recipes)

  75. Lysis buffer (see Recipes)

  76. Wash buffer (see Recipes)

  77. Elution buffer (see Recipes)

  78. IEX A buffer (see Recipes)

  79. IEX B buffer (see Recipes)

  80. Gel-filtration buffer (see Recipes)

  81. 100 mM PMSF (see Recipes)

  82. 10 mg/mL DNase I (from bovine pancreas) (see Recipes)

  83. 0.5 N NaOH + 1 mM EDTA solution (see Recipes)

  84. DEPC-treated H2O (see Recipes)

  85. 10× IVT buffer (see Recipes)

  86. 50% PEG 2000 (see Recipes)

  87. 25 mM NTP mix (see Recipes)

  88. 1 M DTT (see Recipes)

  89. 0.5 M EDTA (see Recipes)

  90. 2× STOP solution (see Recipes)

  91. Urea-PAGE premix (see Recipes)

  92. 20% APS (see Recipes)

  93. 1 mg/mL glycogen (see Recipes)

  94. RNA dissolving buffer (see Recipes)

Equipment

  1. Rainin Pipet-Lite XLS single channel pipettes, 2-μL, 20-μL, 200-μL, and 1,000-μL (Mettler Toledo, catalog numbers: 17014393, 17014392, 17014391, and 17014382)

  2. Thermo Scientific S1 pipet fillers (ThermoFisher, catalog number: 9501)

  3. Autoclave (TOMY, catalog number: SX700)

  4. Vortex mixer (Scientific Industries, catalog number: G560)

  5. AKTA Purifier 10 FPLC system (GE Healthcare, catalog number: 28406264)

  6. Mini-PROTEIN Tetra vertical electrophoresis cell (Bio-Rad, catalog number: 1658004)

  7. SimpliAmp PCR thermal cycler (ThermoFisher, catalog number: A24811)

  8. Mini gel DNA electrophoresis system (ThermoFisher, catalog number: B1)

  9. Owl P10DS vertical electrophoresis system (ThermoFisher, catalog number: P10-DS-1)

  10. Bio-Rad PowerPac HV power supply (Bio-Rad, catalog number: 1645056)

  11. Gel imaging system (Azure Biosystems, catalog number: C300)

  12. Compact UV lamp (UVP, catalog number: 95-0016-14)

  13. Bacterial shaking incubator with refrigerating function (TKS, catalog number: OSI-500R eco)

  14. Cell density meter (WPA, catalog number: C08000)

  15. Sonicator system with 0.5”-diameter probe horn (Qsonica, catalog numbers: Q700 and 4420)

  16. Peristaltic pump (EYELA, catalog number: MP-2000)

  17. Fresco 21 microcentrifuge (ThermoFisher, catalog number: 75002555)

  1. Allegra centrifuge (Beckman-Coulter, catalog number: X-30R)

  2. Avanti centrifuge (Beckman-Coulter, catalog number: J-25)

  3. Centrifuge rotor for 50-mL conical tube (Beckman-Coulter, catalog number: SX440)

  4. Centrifuge rotor for Oak Ridge PPCO tube (Beckman-Coulter, catalog number: JA-25.50)

  5. Centrifuge rotor for 1-L polypropylene bottle (Beckman-Coulter, catalog number: JLA-8.1)

  6. Electronic scale (Mettle Toledo, catalog number: ML3002)

  7. pH meter (Mettle Toledo, catalog number: 51302807)

  8. NanoDrop Lite Spectrophotometer (ThermoFisher, catalog number: ND-LITE-PR)

  9. MilliQ water purification system (Millipore, catalog number: MP0024)

  10. Water bath, set to 37°C and 42°C (FIRSTEK, catalog number: B206)

  11. Heat block, set to 95°C (Major Science, catalog number: MC01N)

  12. Stir plate (Corning, catalog number: PC620D)

  13. 250-mL Erlenmeyer flask (Pyrex, catalog number: 4980)

  14. 2.5-L Ultra-Yield flasks (Thomson, catalog number: 931136-B)

  15. Pyrex 100-mL beaker (DWK Life Sciences, catalog number: 1000/04D)

  16. Pyrex 400-mL glass beaker (DWK Life Sciences, catalog number: 1000/14D)

  17. Pyrex 500-mL glass bottle (DWK Life Sciences, catalog number: 1516/08D)

  18. Pyrex 1-L glass bottle (DWK Life Sciences, catalog number: 1516/10D)

  19. GL45 Screw cap Twin Hose Connector for Pyrex bottles (Fisher Scientific, catalog number: 15193927)

  20. Three-Prong extension clamp stand (Fisher Scientific, catalog number: 057697Q)

  21. Rotary shaker (ARGOS, catalog number: R2200)

  22. SpeedVac vacuum concentrator (EYELA, catalog number: CVE-2100)

  23. Microplate reader (Tecan, catalog number: M1000Pro)

  24. Vacuum pump (Rocker, catalog number: 400)

Software

  1. UNICORN, for AKTA FPLC operation (version 5.31)

  2. GraphPad Prism 9, for statistics and data visualization (version 9.3.1)

  3. ImageJ, for image processing (version 1.53a)

Procedure

English
中文翻译

文章信息

版权信息

© 2022 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Lin, S. W., Nguyen, V. Q. and Lin, S. (2022). Preparation of Cas9 Ribonucleoproteins for Genome Editing. Bio-protocol 12(10): e4420. DOI: 10.21769/BioProtoc.4420.

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分类

生物工程 > 生物医学工程

细胞生物学 > 细胞工程 > CRISPR-cas9

生物化学 > 蛋白质 > 分离和纯化

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