A magnetically guided in vivo base editing system

Abstract

The present invention relates to the field of medical imaging technology, and in particular to an in vivo base editing system using magnetic navigation, comprising: a magnetic navigation module, used to provide a magnetic navigation electromagnetic selection field and a magnetic navigation electromagnetic excitation field for an experimental subject; a receiving signal acquisition module, arranged on the magnetic navigation module, used to receive a magnetic induction signal of the experimental subject in the magnetic navigation module; an in vivo gene editing module, using pcDNA3.1 as a plasmid vector, DNA binding protein Cas9 and cytidine deaminase or IS621mRNA as a base editor, wherein the base editor is used to realize gene expression including a dTtomato sequence, an EPG sequence and an hAPOBE3A sequence; and a control module, which controls the magnetic navigation module and the receiving signal acquisition module through an FPGA board and a PC.

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] Research on biomagnetic induction properties is at the forefront of international science and technology and has important scientific value. In recent years, innovative basic research has discovered that the EPG magnetoresistance gene, driven by electromagnetic fields in vitro in small animal models, has the important function of regulating the opening of cell surface calcium ion channels in vivo. Therefore, by utilizing biomagnetic induction properties and combining 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 was designed to achieve the expression of genes encoding EPG and near-infrared fluorescent proteins, thereby achieving the goal of in vivo base editing and fluorescent tracing in 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, provided on the magnetic navigation module, for receiving the magnetic induction electric signal of the experimental object in the magnetic navigation module;

[0008] The in vivo gene editing module uses 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 tdTomato sequence, EPG sequence and hAPOBEC3A 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 aperture 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 25kHz and a peak value of 20mT 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 sequence as base editors, and adds sgRNA thereto, wherein the DNA-binding protein Cas9 or IS621 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 further 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 placed 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 by the magnetic navigation module.

[0025] According to an embodiment of the present invention, the normalized processing and analysis uses gradient concentration magnetic particle reception signals to draw a normalized curve, and the reception signals are 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 magnetically guided base editing system. The beneficial effects of the present invention are primarily reflected in: By superimposing a spatiotemporally controllable external magnetic field, the present invention enables remote magnetically guided activation of the EPG gene; Furthermore, the present invention utilizes in vivo gene editing, electromagnetic signal acquisition, and microscopic optical imaging methods to monitor the effectiveness of the magnetic navigation system.

[0032] The present invention will utilize electromagnetic effects that can penetrate biological tissues to an unlimited depth to achieve spatiotemporal control of 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 systems immunology research in small animals is provided that integrates in vivo optical imaging, ex vivo cell flow cytometry, and immunofluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A rough configuration diagram of the 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, provided on the magnetic navigation module, for receiving the magnetic induction signal of the experimental object in the magnetic navigation module;

[0039] The in vivo gene editing module uses pcDNA3.1 as a plasmid vector, sgRNA as a specific base editing location, the DNA binding protein Cas9 and cytidine deaminase or IS621 sequence as base editors. The base editors are used to achieve gene expression. The genes contain tdTomato sequences, EPG sequences, and hAPOBEC3A sequences.

[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 aperture 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 25kHz and a peak value of 20mT 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 the magnetic navigation module and the received signal acquisition module.

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

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

[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 same and reverse current, and uses the FPGA board loaded with AD9708 chip to realize voltage signal reception.

[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 sequence as base editors, and adds sgRNA thereto, wherein the DNA-binding protein Cas9 or IS621 sequence 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 further 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 placed 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 by the magnetic navigation module.

[0065] like Figure 2As shown, the method for base editing using the magnetic navigation in vivo base editing system 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 formed a uniform gradient magnetic field of 3 T / m along the imaging aperture as an electromagnetic selection field, providing a gradient magnetic field centered at a zero-magnetic field point. A pair of open Helmholtz coils formed a pulsed or sinusoidal oscillating magnetic field with a frequency of 25 kHz and a peak value of 20 mT 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 of the zero-magnetic field point passing through the target object generated a magnetic induction electrical signal, while the pulsed magnetic excitation of the atomic nuclear spins generated a magnetic induction electrical signal.

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

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

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

[0072] A signal receiving circuit consisting 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 analysis uses the gradient concentration magnetic particle reception signal to draw a normalized curve, and the reception signal is introduced into the processing analysis.

[0076] The present invention uses Litz wire to form a receiving coil to achieve electromagnetic induction, uses a control module composed of a single-chip microcomputer and a PC to start the magnetic navigation system and signal acquisition, and uses a normalization method to achieve 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 are only specific embodiments of the present invention and are 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 scope of protection 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, provided on the magnetic navigation module, for receiving the magnetic induction signal of the experimental object in the magnetic navigation module; The in vivo gene editing module uses pcDNA3.1 as a plasmid vector, sgRNA as a specific base editing location, the DNA binding protein Cas9 and cytidine deaminase or IS621 sequence as a base editor. The base editor is used to achieve gene expression. The gene contains a tdTomato sequence, an EPG sequence, and a hAPOBEC3A sequence; the EPG sequence is shown in 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 in SEQ ID NO. 4; 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 includes: A pair of permanent magnets, used to form a uniform gradient magnetic field of 3 T / m along the imaging aperture 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, wherein 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, wherein 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.

5. The magnetically guided in vivo base editing system according to claim 1, wherein include: The in vivo gene editing module uses the DNA-binding protein Cas9 and cytidine deaminase or IS621 sequence as base editors, and adds sgRNA thereto, wherein the DNA-binding protein Cas9 or IS621 sequence 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 further includes an analysis module, which uses a normalization method to process and analyze the signal received by the magnetic navigation module.

6. The magnetically guided in vivo base editing system according to claim 5, characterized in that The experimental object is arranged in the imaging hole in the center of the magnetic navigation module, and the electromagnetic selection field and the magnetic navigation electromagnetic excitation field are applied to the experimental object by the magnetic navigation module.

7. The magnetically guided in vivo base editing system according to claim 5, characterized in that Normalized processing and analysis A normalized curve is drawn using the magnetic particle reception signals at gradient concentrations, and the reception signals are introduced into processing and analysis.

8. The magnetically guided in vivo base editing system according to claim 5, wherein 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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