In-vivo base editing system for magnetic navigation

Abstract

The invention relates to the technical field of medical imaging, in particular to a magnetic navigation in-vivo basic group editing system, which comprises a magnetic navigation module, a magnetic navigation module, an in-vivo basic group editing module and an in-vivo basic group editing module, and is characterized in that the magnetic navigation module is used for providing a magnetic navigation electromagnetic selection field and a magnetic navigation electromagnetic excitation field; the receiving signal acquisition module is arranged on the magnetic navigation module and is used for receiving the magnetic induction signal of the experimental object in the magnetic navigation module; the in-vivo gene editing module adopts pcDNA3.1 as a plasmid vector, DNA binding protein Cas9 and cytidine deaminase or IS621mRNA as a base editor, and the base editor is used for realizing gene expression and comprises a dTtomato sequence, an EPG sequence and an hAPOBE3A sequence; and the control module is used for controlling the magnetic navigation module and the received signal acquisition module through an FPGA (Field Programmable Gate Array) board and a PC (Personal Computer) terminal.

Description

Technical Field

[0001] The present invention relates to the field of medical imaging technology, and in particular to an in vivo base editing system using magnetic navigation. Background Art

[0002] The research on biological magnetoreceptive properties is at the forefront of the international science and technology field and has important scientific value. In recent years, basic research has found that the EPG magnetoreceptive gene has the important function of regulating the opening of calcium ion channels on the surface of cells in vivo under the drive of electromagnetic fields in small animal models in vitro. Therefore, by utilizing the biological magnetoreceptive properties, combined with the single-base gene editing technology commonly used in cutting-edge research at home and abroad, using Cas9 and cytosine deaminase or IS621 recombinase, an in vivo base editing technology is designed to achieve the expression of coding genes such as EPG and near-infrared fluorescent protein, so as to achieve the goal of in vivo base editing and fluorescent tracing of small animals.

[0003] The EPG magnetic induction gene expression technology established by international cutting-edge research relies on a visible spectrum photoexcitation technology. Its main disadvantages are the limited penetration depth of visible light in biological tissues and the low efficiency of deaminase-mediated in vivo base editing, making it difficult to apply to the study of complex disease models. Summary of the invention

[0004] Based on this, the present invention proposes an in vivo base editing system with magnetic navigation.

[0005] According to a first aspect of the present invention, there is provided a magnetically guided in vivo base editing system, comprising:

[0006] A magnetic navigation module, used to provide an electromagnetic selection field and a magnetic navigation electromagnetic excitation field for the experimental object;

[0007] A receiving signal acquisition module, arranged on the magnetic navigation module, for receiving the magnetic induction electric signal of the experimental object in the magnetic navigation module;

[0008] In vivo gene editing module, using pcDNA3.1 as a plasmid vector, sgRNA as a specific base editing location, DNA binding protein Cas9 and cytidine deaminase or IS621 sequence as base editors, the base editors used to achieve gene expression include dTtomato sequence, EPG sequence and hAPOBE3A sequence;

[0009] The control module controls the magnetic navigation module and the received signal acquisition module through the FPGA board and the PC.

[0010] According to an embodiment of the present invention, the magnetic navigation module includes:

[0011] A pair of permanent magnets, used to form a uniform gradient magnetic field of 3 T / m along the imaging hole direction as an electromagnetic selection field;

[0012] A pair of open Helmholtz coils are used to form a pulsed or sinusoidal oscillating magnetic field with a frequency of 25 kHz and a peak value of 20 mT along the imaging hole direction, which serves as the electromagnetic excitation field for magnetic navigation.

[0013] According to an embodiment of the present invention, the receiving signal acquisition module is a hollow coil composed of Litz wire as a signal acquisition receiving coil.

[0014] According to an embodiment of the present invention, the EPG sequence is shown as SEQ ID NO.1;

[0015] The tdTomato sequence is shown in SEQ ID NO.2;

[0016] The hAPOBEC3A sequence is shown in SEQ ID NO.3;

[0017] The IS621 sequence is shown as SEQ ID NO.4.

[0018] According to an embodiment of the present invention, the sequence of the pcDNA3.1 plasmid is shown in SEQ ID NO.8;

[0019] The sequence of the DNA binding protein Cas9 is shown in SEQ ID NO.9.

[0020] According to an embodiment of the present invention, the present invention includes:

[0021] The in vivo gene editing module uses the DNA binding protein Cas9 and cytidine deaminase or IS621 mRNA sequence as base editors, and adds sgRNA thereto, wherein the DNA binding protein Cas9 or IS621 mRNA and sgRNA are added at a molar ratio of 1:3;

[0022] The in vivo gene editing module further includes an intraperitoneal injection module, which injects the base editor into the experimental subject to perform in vivo gene editing on the experimental subject;

[0023] The control module also includes an analysis module, which uses a normalization method to process and analyze the signal received by the magnetic navigation module.

[0024] According to an embodiment of the present invention, the experimental object is arranged in an imaging hole in the center of a magnetic navigation module, and an electromagnetic selection field and a magnetic navigation electromagnetic excitation field are applied to the experimental object through the magnetic navigation module.

[0025] According to an embodiment of the present invention, the normalized processing and analysis uses the gradient concentration magnetic particle receiving signal to draw a normalized curve, and the receiving signal is introduced into the processing and analysis.

[0026] According to an embodiment of the present invention, the sgRNA comprises sgRNA1:sgRNA2:sgRNA3 in a molar ratio of 1:1:1, wherein

[0027] The sgRNA1 sequence is shown in SEQ ID NO.5;

[0028] The sgRNA2 sequence is shown in SEQ ID NO.6;

[0029] The sgRNA3 sequence is shown in SEQ ID NO.7.

[0030] It can be seen from the above technical solutions that the magnetically guided in vivo base editing system and in vivo base editing method provided by the present invention have the following beneficial effects:

[0031] The present invention provides an in vivo base editing system with magnetic navigation. The beneficial effects of the present invention are mainly reflected in: the present invention can control the external magnetic field by superimposing time and space to achieve remote magnetic navigation activation of EPG genes; in addition, the present invention adopts in vivo gene editing, electromagnetic signal collection and microscopic optical imaging methods to achieve effect monitoring of the magnetic navigation system.

[0032] The present invention will utilize the electromagnetic effect that can penetrate biological tissues to unlimited depth to achieve spatiotemporal controllable magnetic drive and utilize nonlinear voltage response to reconstruct magnetic particle imaging images to achieve the goal of in vivo gene editing effect monitoring; at the same time, utilizing the technical advantages of optical imaging with high sensitivity and high resolution, a new tool for systemic immunology research that integrates in vivo optical imaging of small animals, in vitro cell flow cytometry, and immunofluorescence imaging is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A rough configuration diagram of a magnetic navigation system according to an embodiment of the present invention;

[0034] Figure 2 Schematic diagram of in vivo base editing according to an embodiment of the present invention. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0036] A magnetically guided in vivo base editing system, comprising:

[0037] A magnetic navigation module, used to provide an electromagnetic selection field and a magnetic navigation electromagnetic excitation field for the experimental object;

[0038] A receiving signal acquisition module, arranged on the magnetic navigation module, for receiving a magnetic induction signal of the experimental object in the magnetic navigation module;

[0039] In vivo gene editing module, using pcDNA3.1 as a plasmid vector, sgRNA as a specific base editing location, DNA binding protein Cas9 and cytidine deaminase or IS621 sequence as base editors, the base editors used to achieve gene expression include dTtomato sequence, EPG sequence and hAPOBE3A sequence;

[0040] The control module controls the magnetic navigation module and the received signal acquisition module through the FPGA board and the PC.

[0041] According to an embodiment of the present invention, Figure 1 As shown, the magnetic navigation module includes:

[0042] A pair of permanent magnets, used to form a uniform gradient magnetic field of 3 T / m along the imaging hole direction as an electromagnetic selection field;

[0043] A pair of open Helmholtz coils are used to form a pulsed or sinusoidal oscillating magnetic field with a frequency of 25 kHz and a peak value of 20 mT along the imaging hole direction, which serves as the electromagnetic excitation field for magnetic navigation.

[0044] The receiving coil receives magnetic induction electrical signals rather than magnetic induction signals. The magnetic induction signals are collected using ivis small animal in vivo imaging and confocal microscopy.

[0045] According to an embodiment of the present invention, the receiving signal acquisition module is a hollow coil composed of Litz wire as a signal acquisition receiving coil.

[0046] According to an embodiment of the present invention, an FPGA board and a PC are used to control a magnetic navigation module and a received signal acquisition module.

[0047] According to the embodiment of the present invention, the start-up signal of the magnetic navigation module is transmitted through a digital-to-analog converter and a power amplifier to generate a stable excitation oscillating magnetic field.

[0048] According to an embodiment of the present invention, the start signal of the receiving signal acquisition module starts the filter and the ILA digital signal acquisition migration respectively, thereby realizing the acquisition of real-time electromagnetic signals.

[0049] The present invention starts the magnetic navigation module through the PC control module, realizes the start of different excitation electromagnetic fields including sine, pulse and co-reverse current, and realizes voltage signal reception by using the FPGA board loaded with AD9708 chip.

[0050] According to an embodiment of the present invention, the EPG sequence is shown as SEQ ID NO.1;

[0051] The tdTomato sequence is shown in SEQ ID NO.2;

[0052] The hAPOBEC3A sequence is shown in SEQ ID NO.3;

[0053] The IS621 sequence is shown as SEQ ID NO.4.

[0054] According to an embodiment of the present invention, the sgRNA comprises sgRNA1:sgRNA2:sgRNA3 in a molar ratio of 1:1:1, wherein

[0055] The sgRNA1 sequence is shown in SEQ ID NO.5;

[0056] The sgRNA2 sequence is shown in SEQ ID NO.6;

[0057] The sgRNA3 sequence is shown in SEQ ID NO.7.

[0058] According to an embodiment of the present invention, the sequence of the pcDNA3.1 plasmid is shown in SEQ ID NO.8;

[0059] The sequence of the DNA binding protein Cas9 is shown in SEQ ID NO.9.

[0060] According to an embodiment of the present invention, the present invention includes:

[0061] The in vivo gene editing module uses the DNA binding protein Cas9 and cytidine deaminase or IS621 mRNA sequence as base editors, and adds sgRNA thereto, wherein the DNA binding protein Cas9 or IS621 mRNA and sgRNA are added at a molar ratio of 1:3;

[0062] The in vivo gene editing module further includes an intraperitoneal injection module, which injects the base editor into the experimental subject to perform in vivo gene editing on the experimental subject;

[0063] The control module also includes an analysis module, which uses a normalization method to process and analyze the signal received by the magnetic navigation module.

[0064] According to an embodiment of the present invention, the experimental object is arranged in an imaging hole in the center of a magnetic navigation module, and an electromagnetic selection field and a magnetic navigation electromagnetic excitation field are applied to the experimental object through the magnetic navigation module.

[0065] like Figure 2As shown, the method for base editing using the in vivo base editing system of magnetic navigation comprises the following steps:

[0066] Move the experimental subject into the imaging hole in the center of the magnetic navigation module;

[0067] applying an electromagnetic selection field and a magnetic navigation electromagnetic excitation field to the experimental subject;

[0068] Apobec3 knockout mice were used as experimental subjects. A pair of permanent magnets were used to form a uniform gradient magnetic field of 3T / m along the imaging aperture as an electromagnetic selection field to provide a gradient magnetic field with a point without magnetic field at its center. A pair of open Helmholtz coils were used to form a pulsed or sinusoidal oscillating magnetic field with a peak frequency of 25khz and a peak value of 20mT along the imaging aperture as a magnetic navigation electromagnetic excitation field to achieve magnetic drive of the EPG magnetic induction protein. The sinusoidal magnetic excitation point without magnetic field passed through the target object to generate a magnetic induction electrical signal, and the pulsed magnetic excitation nuclear spin generated a magnetic induction electrical signal.

[0069] A plasmid vector was used to amplify the corresponding mRNA, a DNA binding protein Cas9 and cytidine deaminase or IS621 were used as base editors, and sgRNA was used as a specific base editor for positioning, wherein the molar ratio of Cas9 or IS621 mRNA and sgRNA was 1:3, and the molar ratio of sgRNA was equal.

[0070] The base editor and sgRNA are embedded in lipid nanoparticles, and the base editor is injected into the experimental subject by intraperitoneal injection to perform in vivo gene editing on the experimental subject;

[0071] In the present invention, Cas9 or IS621 mRNA and sgRNA are injected intraperitoneally at a molar ratio of 1:3 to perform in vivo gene editing on Apobec3 knockout mice, and the results are verified by PCR sequencing and microscopic optical imaging.

[0072] A signal receiving circuit composed of a Litz coil, a signal amplifier, a filter and an FPGA is used to receive the electromagnetic induction signal received by the magnetic navigation module.

[0073] The electromagnetic induction signal of gradient magnetic particles is used to draw a normalized curve, and the normalized method is used to process and analyze the signal received by the magnetic navigation module.

[0074] According to an embodiment of the present invention, the signal received by the magnetic navigation module is an electromagnetic induction signal.

[0075] According to an embodiment of the present invention, the normalized processing and analysis uses the gradient concentration magnetic particle receiving signal to draw a normalized curve, and the receiving signal is introduced into the processing and analysis.

[0076] The present invention utilizes Litz wire to form a receiving coil to realize electromagnetic induction, realizes the start of a magnetic navigation system and signal acquisition through a control module composed of a single-chip microcomputer and a PC end, and utilizes a normalization method to realize the reception, processing and analysis of the navigation space-time signal.

[0077] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A magnetically guided in vivo base editing system, characterized in that: include: A magnetic navigation module, used to provide an electromagnetic selection field and a magnetic navigation electromagnetic excitation field for the experimental object; A receiving signal acquisition module, arranged on the magnetic navigation module, for receiving a magnetic induction signal of the experimental object in the magnetic navigation module; In vivo gene editing module, using pcDNA3.1 as a plasmid vector, sgRNA as a specific base editing location, DNA binding protein Cas9 and cytidine deaminase or IS621 sequence as base editors, the base editors used to achieve gene expression include dTtomato sequence, EPG sequence and hAPOBE3A sequence; The control module controls the magnetic navigation module and the received signal acquisition module through the FPGA board and the PC.

2. The magnetically guided in vivo base editing system according to claim 1, characterized in that: The magnetic navigation module comprises: A pair of permanent magnets, used to form a uniform gradient magnetic field of 3 T / m along the imaging hole direction as an electromagnetic selection field; A pair of open Helmholtz coils are used to form a pulsed or sinusoidal oscillating magnetic field with a frequency of 25 kHz and a peak value of 20 mT along the imaging hole direction, which serves as the electromagnetic excitation field for magnetic navigation.

3. The magnetically guided in vivo base editing system according to claim 1, characterized in that: The receiving signal acquisition module is a hollow coil composed of Litz wire as a signal acquisition receiving coil.

4. The magnetically guided in vivo base editing system according to claim 1, characterized in that: The EPG sequence is shown as SEQ ID NO.1; The tdTomato sequence is shown in SEQ ID NO.2; The hAPOBEC3A sequence is shown in SEQ ID NO.3; The IS621 sequence is shown as SEQ ID NO.

4.

5. The magnetically guided in vivo base editing system according to claim 1, characterized in that: The sequence of the pcDNA3.1 plasmid is shown in SEQ ID NO.8; The sequence of the DNA binding protein Cas9 is shown in SEQ ID NO.

9.

6. The magnetically guided in vivo base editing system according to claim 1, characterized in that: include: The in vivo gene editing module uses the DNA binding protein Cas9 and cytidine deaminase or IS621 mRNA sequence as base editors, and adds sgRNA thereto, wherein the DNA binding protein Cas9 or IS621 mRNA and sgRNA are added at a molar ratio of 1:3; The in vivo gene editing module further includes an intraperitoneal injection module, which injects the base editor into the experimental subject to perform in vivo gene editing on the experimental subject; The control module also includes an analysis module, which uses a normalization method to process and analyze the signal received by the magnetic navigation module.

7. The magnetically guided in vivo base editing system according to claim 6, characterized in that: The experimental object is arranged in an imaging hole in the center of a magnetic navigation module, and an electromagnetic selection field and a magnetic navigation electromagnetic excitation field are applied to the experimental object through the magnetic navigation module.

8. The magnetically guided in vivo base editing system according to claim 6, characterized in that: The normalized processing and analysis draws a normalized curve based on the gradient concentration magnetic particle receiving signal, and the receiving signal is introduced into the processing and analysis.

9. The magnetically guided in vivo base editing system according to claim 6, characterized in that: The sgRNA comprises sgRNA1:sgRNA2:sgRNA3 in a molar ratio of 1:1:1, wherein The sgRNA1 sequence is shown in SEQ ID NO.5; The sgRNA2 sequence is shown in SEQ ID NO.6; The sgRNA3 sequence is shown in SEQ ID NO.7.

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