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Biomineralized CRISPR/Cas9 RNPs nanoparticles, preparation method and application thereof in gene editing

A nanoparticle and biomineralization technology, applied in the field of genetic engineering, can solve problems such as off-target effects and DNA residues, and achieve the effect of maintaining self-activity, maintaining activity, and avoiding inactivation

Pending Publication Date: 2019-12-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, after using nanomaterials as carriers to deliver biomacromolecules, DNA residues are prone to occur, and there are certain off-target effects

Method used

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  • Biomineralized CRISPR/Cas9 RNPs nanoparticles, preparation method and application thereof in gene editing
  • Biomineralized CRISPR/Cas9 RNPs nanoparticles, preparation method and application thereof in gene editing
  • Biomineralized CRISPR/Cas9 RNPs nanoparticles, preparation method and application thereof in gene editing

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preparation example Construction

[0034] see figure 1 As shown, the embodiment of the present invention provides a preparation method of biomineralized CRISPR / Cas9 RNPs nanoparticles, comprising the following steps:

[0035] In parts by volume, add 1 part of 60 mM soluble phosphate into 15 to 18 parts of buffer solution and mix evenly. The buffer solution has a pH value of 7 to 8, an osmotic pressure of 150 to 300 mM, and does not contain P and / or Ca; add 4-5 parts of protein / plasmid DNA to be mineralized into the mixture and mix evenly, then add 4-5 parts of soluble calcium salt with a concentration of 100mM, mix evenly and let it stand until the solution is opaque and there are no visible particles. Precipitate, add a dispersant, shake and mix evenly, and centrifuge, take the supernatant to obtain the primary product of mineralized nanoparticle solution loaded with biomacromolecules, purify the primary product, and obtain a purified solution, hereinafter referred to as the macromolecular solution.

[0036] ...

Embodiment 1

[0045] Example 1 Biomineralization of CRISPR / Cas9 RNPs

[0046] To 183ul mineralization buffer (20mM Tris-HCl, 300mM NaCl, pH=7.5), add 11μl concentration of 60mM Na 2 HPO 4 (can be other orthophosphates, such as NaH 2 PO 4 、KH 2 PO 4 etc.) and vortex to mix, add 50ul of RNPs to be mineralized and mix well, then add 25μl of 100mM CaCl 2 (Calcium nitrate can be used) and oscillate to mix, let stand at room temperature for 2-10mins, when the solution becomes opaque and no precipitation visible to the naked eye, add 5ul 1M PAA (Polyacrylic Acid, polyacrylic acid) and oscillate to mix, Centrifuge at 12000rpm for 10min, remove the supernatant, add 100ulddH 2 O (double-distilled water) washed twice to remove unmineralized molecules, and the precipitate, that is, the mineralized nanoparticles, was ultrasonically dispersed in ddH 2 O middle.

[0047] see figure 2 As shown, among them, figure 2 a is a schematic diagram of TEM (transmission electron microscopy) characterizat...

Embodiment 2

[0051] Example 2, Mineralization carrying efficiency and enzyme activity determination of biological macromolecules

[0052] S201. Preparation of mineralized nanoparticles loaded with biomacromolecules

[0053] To 183ul mineralization buffer (20mM Tris-HCl, 300mM NaCl, pH=7.5), add 11μl concentration of 60mM Na 2 HPO 4 And shake to mix, add 50ul Cas9 protein or plasmid DNA required for mineralization and mix, then add 25μlof 100mM CaCl 2 And oscillate to mix, let it stand at room temperature for 2-10mins, when the solution becomes opaque and no precipitation visible to the naked eye, add 5ul 1M PAA, oscillate and mix, centrifuge at 10000rpm for 10min, take the upper Clear liquid, obtain the mineralized nanoparticle solution loaded with biological macromolecules, hereinafter referred to as the macromolecular solution.

[0054] S202, at the same time, in addition to using mineralization buffer to replace CaCl 2 In addition, other processing methods are the same as step S201,...

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Abstract

The invention discloses biomineralized CRISPR / Cas9 RNPs nanoparticles, a preparation method and an application of the biomineralized CRISPR / Cas9 RNPs nanoparticles in gene editing, which relate to thefield of genetic engineering, the method comprises the following steps: adding 1 part of soluble phosphate with the concentration of 60mM into 15-18 parts of a buffer solution and uniformly mixing the materials; adding 4-5 parts of protein / plasmid DNA to-be-mineralized into a mixed solution, uniformly mixing the materials, adding 4-5 parts of soluble calcium salt with the concentration of 100mM,uniformly mixing the materials, performing standing until the solution is opaque and has no macroscopic precipitate, adding a dispersing agent, uniformly oscillating the materials, mixing the materials, and centrifuging and purifying a mixture; wherein the particle size of the nanoparticles is 100 to 200 nm; when the nanoparticles are used for gene editing, the cells are in contact with mineralized CRISPR / Cas9 RNPs nanoparticles. When the nanoparticles are used for gene editing, DNA residue and off-target effect can be avoided.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a biomineralized CRISPR / Cas9 RNPs nanoparticle, a preparation method and its use in gene editing. Background technique [0002] The CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated9) system is a widely used genetic tool nowadays. However, most of the current CRISPR / Cas9 still use traditional DNA as the delivery method. For example, Agrobacterium is mainly used for plants. Mediator or gene gun, mainly use viral vectors such as lentivirus, adenovirus, etc. when targeting animals. [0003] However, DNA as a delivery method can easily lead to genome integration. Cas9 and sgRNA remain in the host genome for a long time and overexpression will lead to off-target cutting. At present, IVTs (in vitro transcription products) are mainly used to deliver Cas9 protein and sgRNA or to assemble Cas9 protein and sgRNA. RNPs (nucleic acid protein complexe...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90
CPCC12N15/113C12N15/902C12N2310/10C12N2310/20Y02A50/30
Inventor 韩鹤友李烁钧宋智勇
Owner HUAZHONG AGRI UNIV
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