CN109971790A - A kind of near infrared light controlling gene edit methods - Google Patents
A kind of near infrared light controlling gene edit methods Download PDFInfo
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Abstract
The invention discloses a kind of methods of the light-operated gene editing of near-infrared.UCNPs-Cas9 complex of the invention is by the UCNPs@SiO after Carboxylation2It covalently connects CRISPR/Cas9 system under the effect of 4- methylol -3- nitrobenzoic acid to obtain, and after wrapping one layer of PEI, which can be by cell endocytic.Later, under Infrared irradiation, up-conversion nanoparticles can launch ultraviolet light, cut off the connection of Cas9 albumen and up-conversion nanoparticles, to release albumen, albumen is made to get enter into nucleus, realize gene editing.Due to the strong penetration into tissue of infrared light, so that the present invention is in vivo applications, it is with the obvious advantage.
Description
Technical field
The invention belongs to field of biotechnology, disclose the method for near infrared light controlling gene editor a kind of.
Background technique
Short palindrome repetitive sequence (CRISPR) associated protein 9 (Cas9) technology of tufted aturegularaintervals is presently most popular
A kind of gene editing tool, it relies on one section of RNA(sgRNA) carry out site identification, by non-homologous end joining (NHEJ) or
Gene mutation is corrected with source orientation reparation (HDR).However, CRISPR/Cas9 system is due to needing to transmit two macromoleculars simultaneously:
Cas9 albumen (about 160 kd) and sgRNA(are more than 100 bp), therefore its delivering is always a problem, especially on space-time
It accurately controls in terms of the delivering of CRISPR/Cas9 system.In recent decades, pressing based on the design of stimuli responsive nano material
Transport system is needed to obtain extensive concern in nanometer medicine.In various control strategies, light regulation is had been demonstrated
It is a kind of ideal non-invasive manner.By light, the release of bioactive molecule can be easily monitored to, and be had very high
Space-time precision.Up to the present, several photosensitive moleculars have been developed, such as azobenzene derivatives, spiropyran derivatives and band
The photosensitive molecular of adjacent nitric acid benzyl group.These molecules are to issue third contact of a total solar or lunar eclipse isomerization or ester bond in the ultraviolet light of high-energy disconnected
It splits.However, ultraviolet biological body penetration capacity is poor and irradiation for a long time easily causes canceration.Therefore, regulated and controled using infrared light
Gene editing has prospect especially.
Summary of the invention
For the purpose of infrared light controlling gene editor, Cas9 system is utilized 4- methylol -3- nitrobenzoyl by the present invention
Acid is covalently attached on up-conversion nanoparticles (UCNPs).Since up-conversion nanoparticles can be in infrared ray excited lower sending
Ultraviolet light, so that the ester linkage breaking that 4- methylol -3- nitrobenzoic acid and nanoparticle surface are formed, to release Cas9 body
System makes its (i.e. Cas9/sgRNA) to enter nucleus, realizes living cells gene editing.
In order to achieve the above-mentioned object of the invention, the scheme of near infrared light controlling gene editor of the present invention is as follows: a kind of
It can be used for the UCNPs-Cas9@PEI complex of infrared light gene editing, the preparation process of the complex are as follows: by nanoparticle
UCNPs@SiO2Surface carry out it is Carboxylation, then by photosensitive molecular by CRISPR/Cas9 system and nanoparticle UCNPs@SiO2
Covalent linkage obtains UCNPs-Cas9 complex, and wherein CRISPR/Cas9 system includes Cas9 albumen and target gene
SgRNA, and under the action of Electrostatic Absorption, positively charged PEI is superscribed in UCNPs-Cas9 complex outermost layer, is obtained
UCNPs-Cas9@PEI complex；It, can be in infrared light tune when UCNPs-Cas9@PEI complex is delivered into cell or mouse
Control is lower to realize gene editing.
The UCNPs@SiO2It is the up-conversion nanoparticles for having wrapped up layer of silicon dioxide, wherein upper conversion nano grain
Son is NaYF4:Yb/Tm。
The molecule that the Carboxylation process uses for N- trimethoxy silicon propyl ethylenediamine triacetic acid trisodium salt (CAS#:
128850-89-5), when carboxylic acid reaction N- trimethoxy silicon propyl ethylenediamine triacetic acid trisodium salt 6 μ g/mL of concentration.
The photosensitive molecular be 4- methylol -3- nitrobenzoic acid (molecular weight: 181.15, CAS#:3113-71-1),
4- methylol -3- nitrobenzoyl acid concentration is 10 μ g/mL when photosensitized reaction.
The Cas9 albumen and sgRNA mass ratio is 1:2.The PEI Chinese is polyethyleneimine (molecule
Amount: 10000), the sgRNA mass ratio with addition is 5:1.
UCNPs@SiO in the UCNPs-Cas9@PEI complex2Final concentration of 3.5 mg/mL, Cas9 albumen are whole
0.36 μ g/mL, sgRNA final concentration of concentration, 0.72 μ g/mL.
In the infrared light regulation process, 980 nm of infrared light wavelength of use, 20 min of light irradiation time.
The present invention also provides a kind of near infrared light controlling gene edit methods, include the following steps:
By UCNPs-Cas9@PEI complex above-mentioned, the editor to target gene is realized under 980 nm Infrared irradiations.Institute
The target gene stated includesEGFP、Plk-1Gene, cell are A549 lung carcinoma cell, and mouse is BALB/c nude mice.
The present invention with 4- methylol -3- nitrobenzoic acid (ONA) by wrapped up silica up-conversion nanoparticles and
CRISPR/Cas9 system is covalently attached, in order to improve endocytosis effect, on nanoparticle (the i.e. UCNPs-Cas9 complex) surface
Wrap up a strata etherimide (Polyetherimide, PEI).By the composite nanoparticle (UCNPs-Cas9@PEI) and cell
It is incubated for altogether, realizes cytogene regulation (Fig. 1) under infrared light regulation.In addition, the system is also applied to living body treatment by us
In.Its specific reaction step is as follows:
(1) preparation of UCNPs-Cas9@PEI
Firstly, in UCNPs@SiO220 μ L N- trimethoxy silicon propyl ethylenediamine triacetic acid trisodium reactant salts 4.5 of middle addition are small
Shi Hou, Carboxylation due to nanoparticle surface, current potential is down to -41.5 mV(Fig. 2 by -28.3 mV).Followed by ONA and
Carboxylation nanoparticle is by the connection of esterification, and wherein solvent uses dry tetrahydrofuran, and catalyst is two hexamethylenes
Base carbodiimide and 4-dimethylaminopyridine.After 12 hours, is washed 3 times with tetrahydrofuran, continuously add dicyclohexyl carbon two
The further activated carboxyl of imines.It then collects particle and is transferred in water phase, due to the connection of surface ONA, current potential occurs again
Variation, becomes -12.7 mV.Meanwhile sgRNA is added with the mass ratio of 1:2 in Cas9 protein solution, solution from positive electricity (+
20.0mV) become negative electricity (- 22.2 mV), illustrates the formation (Fig. 2) of CRISPR/Cas9 system.Then, by CRISPR/Cas9
System is mixed with Carboxylation nanoparticle-ONA, and 4oC is incubated overnight.After centrifugation, in order to improve endocytosis effect, with positively charged
PEI wraps up UCNPS-Cas9, and equilibrium at room temperature after five minutes, measures current potential by -36.1 mV and is changed into 15.0 mV.
(2) release of infrared light control CRISPR/Cas9
It responds, UCNPs-Cas9 solution can be exposed under near-infrared laser (980 nm) in order to verify the near-infrared of system,
Different moments centrifuging and taking supernatant surveys the absorption peak strength at 280 nm (common absorbing proteins peak), as a result as shown in Figure 3A, with
The increase of time, absorption peak enhancing, it means that albumen falls off from particle.In addition, in order to verify up-conversion nanoparticles
Infrared light is converted to the effect of ultraviolet light, it can be by the exposure of UCNPs-Cas9 solution under ultraviolet light, in different moments centrifuging and taking
Supernatant surveys the absorption peak strength at 280 nm (common absorbing proteins peak), obtains similar result such as Fig. 3 B.
(3) cytotoxicity of UCNPs-Cas9@PEI
After the toxicity of nanoparticle UCNPs-Cas9@PEI can be by the way that it to be incubated for lung carcinoma cell (A549) jointly respectively, benefit
The activity that cell under different condition is detected with Cell Counting Kit-8 (CCK-8) detection method, is as a result shown in Fig. 4.From experiment
As a result nanoparticle concentration, infrared luminous intensity and light irradiation time used in cell after being determined in.SgRNA is targeted in this experiment
Site be non-lethality geneEGFP, target sequence are as follows:
5 '-GGGCGAGGAGCTGTTCACCG-3 ' (SEQ ID NO.1).
(4) the infrared light control gene editing in cell
Plk-1Gene is a kind of gene relevant to cancer cell multiplication, can inhibit the growth of cancer cell to the knockout of the gene.
In order to verify the infrared light control gene editing effect of UCNPs-Cas9@PEI, can by UCNPs-Cas9@PEI and cell incubation, 48
Cell pyrolysis liquid is extracted after hour, gene mutation rate is detected by T7 endonuclease I, (Fig. 5 A) and Western
Blot(Fig. 5 B) target point protein is analyzed, it is thus identified that the feasibility of infrared light control gene editing.It is dead to cell from streaming
Analysis living can intuitively confirm infrared light in this system to cells apoptosis (Fig. 5 C).In step (4) and step (5)
In, the site of sgRNA targeting isPlk-1Gene targets sequence are as follows:
5 '-TACCTACGGCAAATTGTGCT-3 ' (SEQ ID NO.2).
(5) therapeutic effect of the infrared light control gene editing for mouse tumor
Tumor (A549 cell) is grown to mouse, reaches 80 mm to tumor size3Afterwards, it is randomly divided into 5 groups, every group 5, and respectively
With PBS+NIR, UCNPs@PEI+NIR, UCNPs@PEI+free Cas9+NIR, UCNPs-Cas9@PEI(without NIR) and
UCNPs-Cas9@PEI+NIR is handled, administration monitoring 20 days.During this period, gross tumor volume is recorded.Finally at the 20th day
To euthanizing animals.Its result is as shown in Figure 6.
It compared with the existing technology, can the invention has the following beneficial effects: (1) present invention utilizes this noninvasive external means of light
It realizes and gene editing is carried out to target position, improve the targeting of gene editing；(2) since the penetration into tissue of infrared light is than purple
It is outer strong, therefore this invention can be very good to apply in biological living.
Detailed description of the invention
Fig. 1 is the reaction principle figure of the method；(A) be composite nanoparticle UCNPs-Cas9@PEI preparation method；
(B) for NIR triggering Cas9-sgRNA into nuclear delivery process: (I) in conjunction with cell membrane, (II) endocytosis, (III) intension
Body escape, (IV) sheds into nucleus from nanoparticle, and (V) finds target dna site and realize that double-strand is cut；
Fig. 2 is the current potential of each process of Nanoparticle Modified；Current potential is successively from left to right Cas9 albumen, Cas9/ in figure
sgRNA, UCNPs@SiO2、UCNPs@SiO2-COOH、 UCNPs@SiO2- ONA, UCNPs-Cas9 and UCNPs-Cas9@
PEI；
Fig. 3 is that the supernatant of UCNPs-Cas9 changes over time figure in the UV absorption of 280 nm, and Fig. 3 A is in Infrared irradiation
Under, Fig. 3 B is under ultraviolet light；
Fig. 4 is the activity for the cell at different conditions that CCK-8 detection method measures, and Fig. 4 A is that investigate is 980 under different capacity
Influence of the nm laser to cell activity, the active influence of concentration versus cell of Fig. 4 B difference nanoparticle, when Fig. 4 C difference illumination
Long influence；
Fig. 5 is the verifying of cellular layer face infrared light control gene knockout, and Fig. 5 A is inspection of the T7 endonuclease I to gene mutation rate
It surveys, Fig. 5 B is after Western blot handles different condition, and the content (A549 cell) of the Plk-1 albumen measured, Fig. 5 C is not
The cell survival rate measured using streaming under the conditions of；I: PBS+NIR, II: CRISPR/Cas9@PEI+NIR, III:
UCNPs+NIR, IV: UCNPs-Cas9@PEI, V: UCNPs-Cas9@PEI+NIR.
Fig. 6 is through various preparations treated tumor growth curve.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment and attached drawing content that the present invention is furture elucidated, but this
The content of invention is not limited solely to following embodiment.Wherein used experimental method is normal unless otherwise specified
Rule method.
Embodiment 1
The present embodiment provides the preparation methods of UCNPs-Cas9@PEI:
UCNPs@SiO2(100 mg) is carried out with 20 μ L N- trimethoxy silicon propyl ethylenediamine triacetic acid trisodium salts it is Carboxylation,
React 4.5 h.Then Carboxylation nanoparticle is transferred to 10 mL(containing 100 μ g ONA) in dry tetrahydrofuran with two hexamethylenes
Base carbodiimide and 4-dimethylaminopyridine are as catalyst progress esterification.After 12 hours, 3 are washed with tetrahydrofuran
It is secondary, then 20 μ g dicyclohexylcarbodiimides are dissolved in tetrahydrofuran, further activated carboxyl.After 8 hours, grain is collected
Son is simultaneously transferred in PBS.Meanwhile mixing Cas9 albumen and sgRNA(1:2) 5 minutes in PBS, form CRISPR/Cas9 body
System.Then, by CRISPR/Cas9 system and Carboxylation nanoparticle-ONA(Carboxylation nanoparticle i.e. above-mentioned and ONA
Connection product) mixing, and 4oC is incubated overnight.After centrifugation, PEI is wrapped in by UCNPS- with 5:1 weight ratio (PEI:sgRNA)
On Cas9, and further balance after five minutes, can be used to cell research at room temperature.The surface modification of this process can benefit
Verifying (Fig. 2) is able to current potential.
Embodiment 2
The present embodiment demonstrates the near-infrared response of system, the method is as follows:
Using collimator, the light intensity of 980 nm near infrared lasers is fixed on 2 W/cm2.By UCNPs-Cas9 solution as this
Under light intensity, supernatant is obtained every 5 min centrifuging and takings, surveys the ultraviolet absorption peak at 280 nm (common absorbing proteins peak), as a result
As shown in Figure 3A, as time increases, absorption peak enhances, it means that albumen falls off from particle.In addition, being to verify
Infrared light is converted to the effect of ultraviolet light by up-conversion nanoparticles, can be by the exposure of UCNPs-Cas9 solution under ultraviolet light, often
Supernatant is obtained every 2.5 min centrifuging and takings and surveys 280 nm absorption peaks, obtains similar result such as Fig. 3 B.
Embodiment 3
Nano-complex (the sgRNA targeting of testing example 1EGFPGene targets sequence are as follows:
5 '-GGGCGAGGAGCTGTTCACCG-3 ' (SEQ ID NO.1)) cytotoxicity, the method is as follows:
It selects lung carcinoma cell (A549), irradiates 10 minutes under the infrared light of varying strength, obtained from CCK-8 detection method respectively
As a result from the point of view of, 0.5 ~ 2.5 W/cm2Infrared light can't have too much influence (Fig. 4 A) to cell activity.Then, 2.0
W/cm2Infrared light intensity under, choose the compound bulk concentration of different UCNPs-Cas9 PEI and cell incubation, find nanoparticle to thin
The toxicity of born of the same parents increases (Fig. 4 B) as concentration increases.Finally, influence of the different light irradiation times for cell activity is investigated, in order to
Reduce UCNPs-Cas9@PEI complex itself with infrared light condition for the toxic effect of cell, in cell experiment later,
It is all made of 2.0 W/cm2Infrared irradiation 20 minutes, and the compound bulk concentration of UCNPs-Cas9 PEI is controlled in 50 ppm.
Embodiment 4
The present embodiment provides the external specific steps for carrying out infrared light control gene editing: preparing and is targeted to according to embodiment 1Plk-1The nano-complex of gene (it is SEQ ID NO.2 that it, which targets sequence), chooses the hypotoxicity condition determined in embodiment 3,
With cell incubation 48 hours.By streaming intuitively to Apoptosis analysis for statistical analysis, as a result such as Fig. 5 C, UCNPs-
The Cas9@PEI+NIR experimental group evening rate of withering reaches 50 %, is much higher than other groups.Then, in order to verify Apoptosis and target gene
Related, extractable cell pyrolysis liquid is knocked out, gene mutation rate is detected by T7 endonuclease I, verifying infrared light regulates and controls lower mesh
The knockout of gene is marked, Fig. 5 B has confirmed in experimental group V:UCNPs-Cas9@PEI+NIR,Plk-1Gene is largely knocked out.This
Outside, target point protein is analyzed by the experiment of Western blot, has also obtained similar result (Fig. 5 A), this is into one
Step analyzes the feasibility of infrared light control gene editing.In order to prevent since 980 nm laser cause culture medium mistake in the present embodiment
The problem of heat should allow cell cooling once every 30 seconds, be further continued for allowing laser irradiation.
Embodiment 5
The present embodiment zoopery is carried out in the case where Nanjing University experimental animal uses and administration committee instructs.This implementation
Example demonstrates the (targeting of infrared light control gene editingPlk-1Gene) effect on mouse living body, specific experiment step:
By tumor-bearing mice (A549, about 80 mm of tumor size3) 5 groups are randomly divided into, every group 5, PBS+NIR, UCNPs@are used respectively
PEI+NIR, UCNPs@PEI+free Cas9+NIR, UCNPs-Cas9@PEI(are without NIR) and UCNPs-Cas9@PEI+NIR
Processing, every 3 days by reagent of intra-tumoral injection, 100 μ L of drug dose, concentration is 3.5 mg/ml.In experimental group
980 nm pulse laser intensity use 2.0 W/cm2, irradiate 25 minutes every time.Administration monitoring 20 days records tumour during this period
Volume is for analysis, then at the 20th day to euthanizing animals.As a result such as Fig. 6, only in UCNPs@PEI+NIR experimental group
Gross tumor volume is controlled in mouse, and gross tumor volume constantly increases in other group of mouse.It means that close red by the present invention
The method of outer smooth controlling gene editor handles (UCNPs@PEI+NIR), can effectively control the growth of tumour, it was demonstrated that successfully complete
Gene editing (target gene knockout).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
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<120>a kind of near infrared light controlling gene edit methods
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<141> 2019-04-01
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tacctacggc aaattgtgct 20
Claims (10)
1. a kind of UCNPs-Cas9@PEI complex that can be used for infrared light gene editing, it is characterised in that:
The preparation process of the complex are as follows: by nanoparticle UCNPs@SiO2Surface progress is Carboxylation, then will by photosensitive molecular
CRISPR/Cas9 system and nanoparticle UCNPs@SiO2Covalent linkage obtains UCNPs-Cas9 complex, wherein CRISPR/
Cas9 system includes the sgRNA of Cas9 albumen and target gene, and under the action of Electrostatic Absorption, in UCNPs-Cas9 complex
Outermost layer superscribes positively charged PEI, obtains UCNPs-Cas9@PEI complex；It is delivered when UCNPs-Cas9@PEI complex
Into cell or mouse, gene editing can be realized under infrared light regulation.
2. UCNPs-Cas9@PEI complex according to claim 1, it is characterised in that:
The UCNPs@SiO2It is the up-conversion nanoparticles for having wrapped up layer of silicon dioxide, wherein up-conversion nanoparticles are
NaYF4:Yb/Tm。
3. UCNPs-Cas9@PEI complex according to claim 1, it is characterised in that:
The molecule that the Carboxylation process uses is N- trimethoxy silicon propyl ethylenediamine triacetic acid trisodium salt, carboxylic acid reaction
When N- trimethoxy silicon propyl ethylenediamine triacetic acid trisodium salt 6 μ g/mL of concentration.
4. UCNPs-Cas9@PEI complex according to claim 1, it is characterised in that:
The photosensitive molecular is 4- methylol -3- nitrobenzoic acid, 4- methylol -3- nitrobenzoyl acid concentration when photosensitized reaction
For 10 μ g/mL.
5. UCNPs-Cas9@PEI complex according to claim 1, it is characterised in that:
The Cas9 albumen and sgRNA mass ratio is 1:2.
6. UCNPs-Cas9@PEI complex according to claim 1, it is characterised in that:
The PEI Chinese is polyethyleneimine, and the sgRNA mass ratio of PEI and addition is 5:1.
7. UCNPs-Cas9@PEI complex according to claim 1, it is characterised in that:
UCNPs@SiO in the UCNPs-Cas9@PEI complex2Final concentration of 3.5 mg/mL, Cas9 final concentration of protein
0.36 μ g/mL, sgRNA final concentration, 0.72 μ g/mL.
8. UCNPs-Cas9@PEI complex according to claim 1, it is characterised in that:
In the infrared light regulation process, 980 nm of infrared light wavelength of use, 20 min of light irradiation time.
9. a kind of near infrared light controlling gene edit methods, which comprises the steps of:
By the described in any item UCNPs-Cas9@PEI complexs of claim 1 ~ 8, the realization pair under 980 nm Infrared irradiations
The editor of target gene.
10. according to the method described in claim 9, it is characterized in that, target gene includesEGFP、Plk-1Gene, cell are
A549 lung carcinoma cell, mouse are BALB/c nude mice.
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