WO2019120046A1 - Method utilizing crispri gene knockdown technique for quick construction of acute cerebral scientific research model - Google Patents

Abstract

Provided are uses of sgRNA/CRISPRi for genetic modification in non-dividing neuronal cells. Also provided are a method for constructing a gene inactivation model, a primary neuron and an animal model constructed per the method.

Description

Method for rapidly constructing an acute brain science research model using CRISPRi gene knockdown technology

Priority information

The present application claims priority to and the benefit of the patent application Serial No. PCT Application No.

Technical field

The present invention relates to the field of gene editing, and in particular, to the use of sgRNA/CRISPRi for genetic engineering in non-dividing neuronal cells, and methods for rapidly constructing cell and animal models of neurobiological research.

Background technique

The CRISPR/Cas9 system from bacteria and archaea has been developed as a genome editing tool and is widely used to establish transgenic cell lines and animal lines. Under the guidance of the guide RNA (sgRNA), Cas9 recognizes a specific target genomic region and introduces a double-strand break gap (DSB), and then the cells repair the gap mainly by random repair. Achieving precise genetic manipulation is currently largely dependent on the transgenic animal operating platform, the efficiency of gene knockout/knock-in and the lengthy production cycle still limit the wider use of genetically engineered animals in the laboratory. Gene editing technology based on the development of various virus tools enables the in vivo manipulation, from molecular to cellular, from loop to behavior, to resolve genotype/phenotype associations at various stages after birth, and under physiological and pathological conditions. Explore the pathogenesis of brain diseases and seek new and effective treatment strategies based on them.

In addition to the fact that a single gene may play a crucial role in biological processes, many life processes require multiple genes or signaling pathways to work together. To date, the interpretation of multi-gene complex psychiatric disorders and the dynamic function of protein complexes at the cellular and animal levels remains a major challenge in biological research, which is of great significance for understanding new patterns of disease progression and protein interaction.

The brain is made up of many different types of neurons that play different roles in life and in the progression of neurological diseases. For example, dopaminergic, serotoninergic, small albumin and somatostatin can be closely related to mental disorders and neurodegenerative diseases. Interfering neuronal activity is an important method for studying specific neuronal subpopulations and the loop/behavior variants associated with them. Thus, flexible gene interference tools for specific neuronal subpopulations will help to elucidate patterns of gene-neuron subpopulation-loop-behavior.

There is still a need in the art to develop simple and easy gene interference tools to obtain single or multiple gene knockdown, as well as research models for gene inactivation of cells or animals of a particular subset of neurons.

Summary of the invention

This application is based on the discovery of the following problems or facts by the inventors:

On the one hand, the inventors found that due to the random repair of DNA by neurons, a variety of genotype/phenotypic changes may occur at the level of a single neuron, such as wild type, chimeric, completely inactive, or even functional. Acquired and so on. Moreover, terminally differentiated neurons have non-separable characteristics, and thus it is difficult for neuronal cells to establish a genotype-like cell line like a dividing cell for research use.

On the other hand, in the prior art, the method of obtaining multiplexed gene-inactivated neurons or animal models by using mouse strains of different strains is not only time-consuming and research-funded, but also lacks flexible variability targeting any multiplex gene combination. Multiple post-inactivated cells or animal models can be obtained by directly injecting a mouse carrying a CRISPR/Cas9 gene editing system into a mouse after birth, but multiple genes targeted by them often exhibit multiple chimeric combinations inactivation, and they The proportion of all simultaneously inactivated neurons is generally no more than 20%, so the pattern of inactivation of such multiple gene chimeric combinations is highly likely to produce a confusing phenotype at the single cell level.

The inventors have unexpectedly discovered through experiments that sgRNA/CRISPRi can improve phenotypic uniformity at a single neuron level and exhibit superior gene silencing of superior targeted genes in neurons.

The present invention aims to solve at least one of the technical problems in the related art to some extent.

To this end, in a first aspect of the invention, the invention proposes the use of sgRNA/CRISPRi for genetic engineering in non-dividing cells. The inventors have found that sgRNA/CRISPRi can prevent targeted gene transcription initiation in non-dividing cells, increase phenotypic uniformity at the individual cell level, and exhibit superior expression-specific gene silencing in non-dividing cells.

According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:

According to an embodiment of the invention, the non-dividing cells are neuronal cells. With the sgRNA/CRISPRi method according to an embodiment of the present invention, the phenotype at a single neuron level is more uniform, and a more specific gene silencing of a targeted gene is presented in the neuron.

According to an embodiment of the invention, the neuronal cell is an excitatory glutamatergic neuron or an inhibitory interneuron. The expression regulation of a targeted gene in a subset of neurons can be achieved using the sgRNA/CRISPRi method according to an embodiment of the present invention.

According to an embodiment of the present invention, the genetic modification is achieved by introducing an endotoxin-free sgRNA/CRISPRi expression plasmid with pVSVG, psPAX2 into a recipient cell to obtain a lentivirus carrying sgRNA/CRISPRi, sgRNA/CRISPRi is a sgRNA/CRISPRi targeting a gene; and the neuronal cell is infected with the lentivirus. This prevents the initiation of transcription of the targeted gene.

According to an embodiment of the invention, the recipient cell is a HEK 293FT cell.

According to an embodiment of the invention, the introduction is PEI mediated.

According to an embodiment of the invention, the gene is a gene expressed in a neuron or a foreign gene. According to a particular embodiment of the invention, the gene may be selected from at least one of Syt1, Vamp2, Stx1a, Snap25, Stx1b, Doc2a, Doc2b, and DsRed.

According to an embodiment of the invention, the sgRNA targets a gene near the transcription initiation region.

According to a particular embodiment of the invention, the sgRNA targets upstream from -50 bp to 300 bp downstream of the transcription initiation region of the gene. Furthermore, sgRNA can target a single protein-encoding gene in a neuron, prevent the transcription initiation of a targeted gene, and cause down-regulation of gene expression, thereby performing gene functional identification.

According to an embodiment of the present invention, the sgRNA has the nucleotide sequences shown in SEQ ID NOS: 1 to 15. The sgRNA having the nucleotide sequences shown in SEQ ID NOS: 1 to 15 targets Syt1, Vamp2, Stx1a, Snap25, Stx1b, Doc2a, Doc2b, and DsRed, and prevents the expression of the above genes.

According to an embodiment of the invention, the sgRNA/CRISPRi expression plasmid is obtained by: (1) providing an initial lentiviral plasmid comprising a human U6 promoter, a stuffer sequence, sgRNA backbone sequence, polymerase type III transcription terminator, EFS promoter, dCas9-KRAB, nuclear localization signal, P2A, EGFP and WPRE nucleic acid sequence elements; (2) insertion of the annealed sgRNA into the initial digestion of BsmbI digestion Lentiviral plasmids were used to obtain a single sgRNA/CRISPRi expression plasmid. Furthermore, sgRNA can target a single protein-encoding gene in a neuron, causing down-regulation of gene expression for gene functional identification.

According to an embodiment of the invention, the sgRNA/CRISPRi expression plasmid is obtained by: (1) providing an initial lentiviral plasmid comprising a sequentially linked human U6 promoter, a stuffer sequence, a sgRNA backbone sequence, a polymerase type III transcriptional terminator, an EFS promoter, a dCas9-KRAB, a nuclear localization signal, a P2A, EGFP, and a WPRE nucleic acid sequence element; (2) a template plasmid comprising a first sgRNA backbone a sequence, a polymerase type III transcription terminator and a human H1 promoter nucleic acid sequence element; (3) amplifying the template plasmid by PCR, wherein the PCR forward primer 3' end comprises the first sgRNA backbone sequence of 5 a 'end sequence, the PCR forward primer has a BsmbI recognition sequence at the 5' end and a first sgRNA sequence to be inserted, and the 3' end of the PCR reverse primer includes the 3' end sequence of the human H1 promoter sequence, the PCR The 5' end of the reverse primer has a reverse complement sequence inserted into the second sgRNA and a BsmbI recognition sequence; (4) after the PCR amplification product in (3) is digested with BsmbI, and ligated to the initial lentiviral plasmid, To get two sgRNA/CRISPRi Body expression plasmid. It should be noted that the "skeletal sequence" described in the present application is a tracrRNA portion, which provides a scaffold support for the action of the crRNA, and is generally an invariant sequence; the "first or second sgRNA sequence" described herein is a target. A 20 bp sequence to the gene of interest, namely crRNA. Furthermore, the two sgRNAs can respectively target two functionally related genes, and simultaneously knock down the two genes in the cell, thereby functionally identifying the cooperation of multiple genes or complex protein machines.

According to a particular embodiment of the invention, the PCR forward primer and reverse primer have the nucleotide sequence set forth in SEQ ID NO: 16 or 17.

According to an embodiment of the invention, the polymerase type III transcription terminator and the human H1 promoter nucleic acid sequence element are tightly linked to reduce the lentiviral vector capacity.

According to an embodiment of the invention, the sgRNA/CRISPRi expression plasmid is obtained by: (1) providing an initial lentiviral plasmid comprising a sequentially linked human U6 promoter, a stuffer sequence, a three sgRNA backbone sequence, a polymerase type III transcriptional terminator, an EFS promoter, a dCas9-KRAB, a nuclear localization signal, a P2A, EGFP, and a WPRE nucleic acid sequence element; (2) a template plasmid comprising a first sgRNA backbone a sequence, a polymerase type III transcription terminator, a human 7SK promoter, a second sgRNA sequence insertion site, the second sgRNA sequence insertion site comprising a BbsI recognition sequence, a second sgRNA backbone sequence, and a polymerase type III transcription termination a sub- and murine U6 promoter; (3) inserting the annealed second sgRNA sequence into the BbsI-digested template plasmid to obtain a template plasmid containing the second sgRNA sequence; (4) the content obtained by PCR amplification a template plasmid of the second sgRNA sequence; wherein the 3' end of the PCR forward primer is a 5' end sequence of the first sgRNA backbone sequence, and the 5' end of the PCR forward primer has a BsmbI recognition sequence and a first s a gRNA sequence, the 3' end of the PCR reverse primer comprising the 3' end of the murine U6 promoter, the 5' end of the PCR reverse primer having a reverse complement of the third sgRNA and a BsmbI recognition sequence; After the PCR amplification product in (4) was digested with BsmbI, it was ligated to the initial lentiviral plasmid to obtain three sgRNA/CRISPRi integrated expression plasmids. It should be noted that the "integrated expression plasmid" as used in the present application means that two or three different pol III promoters drive different sgRNA expressions, and in the same vector as dCas9-KRAB, the gene knock can be directly realized. low. Furthermore, three sgRNAs can be targeted to three functionally related genes, and three genes can be knocked down simultaneously in the cell, thereby functionally identifying the cooperation of multiple genes or complex protein machines.

According to a particular embodiment of the invention, the PCR forward primer and reverse primer have the nucleotide sequence set forth in SEQ ID NO: 20 or 21.

According to an embodiment of the invention, the polymerase type III transcriptional terminator is tightly linked to a human 7SK promoter and a murine U6 promoter. To reduce the capacity of the lentiviral vector.

According to an embodiment of the invention, the sgRNA/CRISPRi binary expression plasmid is obtained by: (1) providing a first initial lentiviral plasmid comprising a sequentially linked EFS promoter, dCas9-KRAB, Nuclear localization signal, P2A, EGFP and WPRE nucleic acid sequence elements; (2) providing a second initial lentiviral plasmid, constructing a single sgRNA/CRISPRi expression plasmid according to the method defined above; (3) amplifying the sgRNA expression cassette by PCR, The sgRNA expression cassette includes a spacer sequence, a human U6 promoter, an sgRNA sequence, an sgRNA backbone sequence, and a polymerase type III transcription terminator; (4) assembling a plurality of sgRNA expression cassettes to the second initial slow using the Golden Gate cloning method The viral plasmid was obtained as a binary expression plasmid of sgRNA/CRISPRi. It should be noted that the "binary expression plasmid" as used in the present application means that a plurality of tandem U6 promoters drive different sgRNA expressions, and dCas9-KRAB is on different vectors, and both must be used together to achieve gene knocking. low. Furthermore, multiple sgRNAs can be targeted to multiple functionally related genes, and multiple genes can be knocked down simultaneously in the cell, thereby functionally identifying the cooperation of multiple genes or complex protein machines.

According to an embodiment of the invention, there are spacer sequences between adjacent sgRNA expression cassettes to reduce mutual transcriptional interference;

According to an embodiment of the invention, the spacer sequence is a lentiviral cPPT (Central Polypurine tract) element. On the one hand, it can increase transduction efficiency and transgene expression; on the other hand, it can reduce transcriptional interference between multiple U6 promoters.

According to a specific embodiment of the present invention, the PCR amplification forward primer and reverse primer have the nucleotide sequences shown in SEQ ID NOS: 22 to 31.

In a second aspect of the invention, the invention proposes a method of constructing a gene inactivation model. According to an embodiment of the invention, genetic modification is performed in non-dividing cells using sgRNA/CRISPR to obtain the gene inactivation model. A method according to an embodiment of the present invention can obtain a model in which a specific gene is inactivated and a phenotype is uniform in non-dividing cells.

According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

According to an embodiment of the invention, the sgRNA/CRISPRi is as previously described.

According to an embodiment of the invention, the non-dividing cells are neuronal cells. A model in which specific genes are inactivated and phenotypically uniform can be obtained in neuronal cells using the method according to an embodiment of the present invention.

According to an embodiment of the invention, the neuronal cell is an excitatory glutamatergic neuron or an inhibitory interneuron. A model in which a specific gene is inactivated and phenotypically uniform can be obtained in a specific subset of neuronal cells using a method according to an embodiment of the present invention.

According to an embodiment of the invention, the gene inactivation model is a cell model or an animal model. The method according to an embodiment of the present invention can inject the single, two, three or more sgRNA/CRISPRi expression plasmids described above into the brain of an animal such as a mouse by means of injection, thereby being effective A model of brain gene inactivity that is inactivated and phenotypically specific for a particular gene.

In a third aspect of the invention, the invention proposes a gene inactivation model. According to an embodiment of the invention, the gene inactivation model is a cell model, and the gene inactivation model is obtained by constructing the method described above. According to the gene inactivation cell model of the present invention, the phenotype is uniform, and gene inactivation has the characteristics of high efficiency and specificity.

It should be noted that the "sgRNA/CRISPRi" described in the present application is a collective name of the sgRNA and CRISPRi technologies. Among them, sgRNA (single guide RNA), also known as "gRNA", is a single-stranded guide RNA, which is a fusion of crRNA and tracrRNA into an RNA strand. CRISPRi (CRISPR) technology refers to the nuclease-free dCas9 and transcriptional repressor KRAB chimera, which bind to the start of transcription of the gene under the guidance of sgRNA and ultimately inhibit the transcription of the targeted gene mRNA.

The additional aspects and advantages of the invention will be set forth in part in the description which follows.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

1 is a flow diagram of a method of constructing a single or multiple sgRNA co-expression plasmids, in accordance with an embodiment of the present invention,

Where a is a flow diagram of a method for constructing an integrated expression plasmid for a single sgRNA,

b is a flow diagram of a method for constructing an integrated expression plasmid for two sgRNAs;

c is a flow diagram of a method for constructing an integrated expression plasmid for three sgRNAs;

2 is a flow diagram of a method for constructing a binary expression plasmid for a plurality of sgRNAs, in accordance with an embodiment of the present invention;

3 is a graph showing the results of CRISPRi efficiently inhibiting the expression of a targeted gene in a primary neuron according to an embodiment of the present invention,

Among them, (a), the principle difference between the CRISPRi gene knockdown technique and the CRISPR gene knockout technique is indicated; the structure of the CRISPRi lentiviral plasmid used by the neurons in the present invention is shown below. P2A-cleaved peptides release dCas9-KRAB and EGFP after auto-splicing, and EGFP can indicate lentivirus-infected neurons.

(b, c) Immunoblotting (b) and semi-quantitative analysis (c) Relative expression levels of proteins encoded by targeting genes in neurons infected with CRISPRi or shRNA/RNAi system lentivirus. Data are shown as mean ± standard error, n = 3;

(d) qRT-PCR semi-quantitative analysis of relative expression levels of mRNA transcribed by targeted genes in neurons infected with CRISPRi or shRNA/RNAi system lentivirus. Data are shown as mean ± standard error, n = 3;

(e, f) Representative trajectories (e) and EPSC amplitudes of excitatory postsynaptic current (EPSC) induced by electrophysiological recordings of action potentials from wild-type, CRISPRi knockdown and gene-suppressed neurons (f). Quantitative data is presented as a box-and-whisker chart. The top and bottom must represent the maximum and minimum values, respectively. The boxes represent 2.5%, 50% and 97.5% quantiles. Unpaired Student's t-test, *P<0.05; **P<0.001.

4 is a graph showing the results of CRISPR-expressing expression-specific gene-specific expression silencing in neurons according to an embodiment of the present invention,

Among them, (a, b) time-tracked immunoblotting (a) and semi-quantitative analysis (b) relative expression levels of Syt1 in neurons infected with CRISPR/shRNA/RNAi lentivirus targeting Syt1 gene;

(c) Time-tracked ChIP-qPCR analysis of the binding kinetics of dCas9-KRAB and dCas9 targeting the Syt1 locus;

(d) Design sgRNA mutants targeting the Syt1 gene to test their ability to bind to the "wrong" Syt1 gene. Blue nucleotides represent mismatch sites in sgRNA;

(e) ChIP-qPCR analysis of the ability of Syt1 sgRNA mutants to direct dCas9-KRAB to target the Syt1 gene;

(f) Semi-quantitative analysis of the effect of Syt1 sgRNA mutants on the relative expression levels of Syt1 gene transcribed mRNA by qRT-PCR;

(g) Immunoblot and semi-quantitative analysis of the effect of Syt1 sgRNA mutants on the relative expression levels of the protein encoded by the Syt1 gene; data are shown as mean ± standard error, n = 3; unpaired Student's t-test, *P<0.05; **P<0.001.

(h) Differential expression analysis The specificity of CRISPRi inactivation of exogenous DsRed gene expression in neurons. RNA-seq data were derived from two separate sets of biological replicates.

5 is a graph showing the results of a conditional inactivation of Syt1 based on CRISPRi, which can alter the excitatory/inhibitory balance of the hippocampal dentate gyrus of mice, according to an embodiment of the present invention,

Among them, (a) illustrates the composition of a neuron subtype-specific CRISPRi conditional inactivation system. The mouse CaMKIIα promoter (pCaMKIIα) and VGAT promoter (pVGAT) drive the expression of dCas9-KRAB in glutamatergic and GABAergic neurons, respectively;

(b) Immunofluorescence representation of the hippocampal dentate gyrus of mice infected with CRISPRi lentivirus. EGFP indicates efficient expression of dCas9-KRAB in infected neurons. Scale bar, 1mm;

(c) A flow chart illustrating the isolation and purification of neurons expressing dCas9-KRAB from mouse brain;

(d) qRT-PCR semi-quantitative analysis of mRNA expression levels of Vglut1 and Gad1 in sorted pCaMKIIα and pVGAT-driven neurons. Data are shown as mean ± standard error, n = 3;

(e) Relative expression levels of Syt1 mRNA in sorted pCaMKIIα and pVGAT-driven neurons. Data are shown as mean ± standard error, n = 3;

(f) Immunoblot analysis of selective inactivation of Syt1 in sorted pCaMKIIα (top) and pVGAT (bottom) driven dCas9-KRAB expressing neurons;

(g) In pCaMKIIα-driven neurons expressing dCas9-KRAB, electrophysiological recording of the control tract, CRISPRi-mediated Syt1 gene knockdown and exogenous Syt1 gene rescued neurons were represented by action potential-induced EPSC (top left) And the EPSC amplitude (bottom left) and the representative trajectory of the inhibitory postsynaptic current (IPSC) recorded in non-lentivirus-infected neurons (top right) and IPSC amplitude (bottom right). EPSC, n=12-30; IPSC, n=10-11. Quantitative data is presented as a box-and-whisker chart. The top and bottom must represent the maximum and minimum values respectively, and the boxes represent 2.5%, 50% and 97.5% quantiles;

(h) In pVGAT-driven expression of dCas9-KRAB neurons, electrophysiological recording of control, CRISPRi-mediated Syt1 gene knockdown and exogenous Syt1 gene rescued neurons by action potential-induced representative path of IPSC (top left) And IPSC amplitude (bottom left) and representative trajectory (upper right) and EPSC amplitude (bottom right) of EPSC recorded in non-infected neurons. IPSC, n=13-35; EPSC, n=10-14. Quantitative data is presented as a box-and-whisker chart. The top and bottom must represent the maximum and minimum values, respectively. The boxes represent 2.5%, 50% and 97.5% quantiles. Unpaired Student's t-test, *P<0.05; **P<0.001.

6 is a graph showing the results of different effects of excitability/inhibition balance of tuned mouse hippocampal dentate gyrus on learning and emotion according to an embodiment of the present invention,

Among them, (a) in the eight consecutive days of water maze (MWM) training, the mouse finds the average delay time of the latent platform;

(b) representative trajectory of the mouse in the water maze test (left) and the mouse in the area where the original latent platform is located and the corresponding target quadrant residence time (right);

(c) a representative trajectory of the mouse in the Barnes Maze Test (BM) (left) and the delay time for the mouse to locate the original hole during the test phase and locate the new hole during the exploration phase;

(d) the percentage of mice correctly alternating in the reward T-maze test (TM);

(e) Percentage of stagnation response in mice in association, alteration, and auditory fear memory tests (FC);

(f) representative trajectories of mice in the open field experiment (OFT) and residence time in the central region;

(g) the open arm residence time of the mouse in the elevated plus maze (EPMT);

(h) the resting time of the mouse in the tail suspension experiment (TST);

(i) The resting time of the mice in the forced swimming test (FST). Data are presented as mean ± standard error, n = 8-10; unpaired Student's t-test, *P < 0.05; ** P < 0.001.

Figure 7 is a graph showing the results of a flexible and diverse CRISPRi multiplex gene knockdown strategy in mouse brain, in accordance with an embodiment of the present invention,

Among them, (a) shows the integrated and dual-CRISPRi vectors;

(b) qRT-PCR semi-quantitative analysis of mRNA relative expression levels of targeted gene transcription in multiplexed gene-targeted CRISPRi lentivirus-infected primary neurons;

(c, d) immunoblot (c) and semi-quantitative analysis (d) relative expression levels of the protein encoded by the targeting gene in multiplex gene-targeted CRISPRi lentivirus-infected primary neurons;

(e) qRT-PCR semi-quantitative analysis of mRNA-relative expression levels of targeted gene transcription in multiplex brain-targeted CRISPRi lentivirus infection in vivo mouse brain neurons. Data are presented as mean ± standard error, n = 3; unpaired Student's t-test, *P < 0.05; ** P < 0.001.

Detailed description of the invention

Embodiments of the invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are intended to be illustrative of the invention and are not to be construed as limiting.

The present invention provides a method of constructing a single or multiple sgRNA co-expression plasmids, which are packaged with dCas9-KRAB into a lentivirus and infect primary neurons or directly injected into the mouse brain to prevent transcription of the targeted gene. beginning. In particular, the designed sgRNA should be targeted near the transcriptional initiation region of the gene, typically from about -50 bp upstream to about 300 bp downstream. For example, in the present invention, sgRNA can target a single protein-encoding gene in a neuron, causing down-regulation of gene expression, thereby performing gene functional identification. For another example, in the present invention, two or more sgRNAs can respectively target two or more functionally related genes, and simultaneously knock down two or more genes in a cell, thereby multiplexing multiple genes or complex proteins. The machine cooperates for functional identification. The present invention also provides a method for expressing dCas9-KRAB in a specific neuronal subpopulation, which drives dCas9-KRAB expression by a neuron subtype-specific promoter, thereby down-regulating the expression of a targeted gene in a corresponding neuron subpopulation, such as excitement. Sexual glutamatergic neurons and inhibitory interneurons.

According to one aspect of the invention, methods of constructing a single or multiple sgRNA co-expression plasmids are provided.

According to a specific embodiment of the invention, referring to Figure 1a, a method for constructing an integrated expression plasmid for a single sgRNA, comprising:

(1) Providing an initial lentiviral plasmid, which comprises a human U6 promoter, a stuffer sequence, a sgRNA backbone sequence, a polymerase type III transcription terminator, an EFS promoter, dCas9-KRAB, Nucleic acid sequence elements such as nuclear localization signals, P2A, EGFP, and WPRE.

(2) An integrated expression plasmid of a single sgRNA was obtained by inserting the annealed sgRNA sequence into a type II (Type IIs) restriction endonuclease BsmbI-digested initial lentiviral plasmid.

According to a specific embodiment of the invention, referring to Figure lb, a method for constructing an integrated expression plasmid for two sgRNAs, comprising:

(3) providing an initial lentiviral plasmid, the first lentiviral plasmid comprising a first human U6 promoter, a stuffer sequence, a second sgRNA backbone sequence, a polymerase type III transcription terminator, an EFS promoter, Nucleic acid sequence elements such as dCas9-KRAB, nuclear localization signals, P2A, EGFP, and WPRE.

(4) A template plasmid 2 is provided, the template plasmid comprising a first sgRNA backbone sequence, a polymerase type III transcription terminator, and a second human H1 promoter.

(5) The template plasmid 2 is amplified by PCR, and the amplified sequence includes a first sgRNA backbone sequence, a polymerase type III transcription terminator, and a second human H1 promoter sequence. Wherein the 3' end of the PCR forward primer is the 5' end portion of the first sgRNA backbone sequence, and the BsmbI recognition sequence and the first sgRNA sequence are added at the 5' end thereof, and the 3' end of the PCR reverse primer is a 3'-end partial sequence of the human H1 promoter sequence, and a reverse complement of the second sgRNA and a BsmbI recognition sequence at its 5' end;

(6) The PCR amplification sequence in (5) was digested with type II restriction endonuclease BsmbI, and ligated with the initial lentiviral plasmid to obtain an integrated expression plasmid of two sgRNAs.

According to a specific embodiment of the invention, referring to Figure 1c, a method for constructing an integrated expression plasmid for three sgRNAs, comprising:

(7) providing an initial lentiviral plasmid, the first lentiviral plasmid comprising a first human U6 promoter, a stuffer sequence, a third sgRNA backbone sequence, a polymerase type III transcription terminator, an EFS promoter, Nucleic acid sequence elements such as dCas9-KRAB, nuclear localization signals, P2A, EGFP, and WPRE.

(8) Providing a template plasmid 3, the template plasmid comprising a first sgRNA backbone sequence, a polymerase type III transcription terminator, a second human 7SK promoter, and a second sgRNA sequence insertion site (including a type II restriction nucleic acid) The Dicer BbsI recognition sequence), the second sgRNA backbone sequence, the polymerase type III transcription terminator, and the third murine U6 promoter.

(9) A template plasmid three containing the second sgRNA sequence was obtained by inserting the annealed second sgRNA sequence into the template plasmid 3 digested with the type II restriction endonuclease BbsI.

(10) Amplification of a template plasmid 3 containing a second sgRNA sequence in (9), the amplified sequence comprising a first sgRNA backbone sequence, a polymerase type III transcription terminator, a second human 7SK promoter, a second sgRNA Sequence, second sgRNA backbone sequence, polymerase type III transcription terminator, third mouse source U6 promoter. Wherein the 3' end of the PCR forward primer is the 5' end portion of the first sgRNA backbone sequence, and the BsmbI recognition sequence and the first sgRNA sequence are added at the 5' end thereof, and the 3' end of the PCR reverse primer is a 3'-end partial sequence of the murine U6 promoter sequence, and a reverse complement of the third sgRNA and a BsmbI recognition sequence at its 5' end;

(11) The PCR amplified sequence in (10) was digested with type II restriction endonuclease BsmbI, and ligated with the initial lentiviral plasmid to obtain an integrated expression plasmid of three sgRNAs.

According to a specific embodiment of the present invention, with reference to Figure 2, a method for constructing a binary expression plasmid for a plurality of sgRNAs, comprising:

(12) An initial lentiviral plasmid IV comprising a nucleic acid sequence element such as a sequence-ligated EFS promoter, dCas9-KRAB, nuclear localization signal, P2A, EGFP and WPRE, i.e., dCas9-KRAB expression plasmid, is provided.

(13) Providing an initial lentiviral plasmid V, constructing an integrated expression plasmid of a single sgRNA according to (1) and (2), and then amplifying the spacer sequence, human U6 promoter, sgRNA sequence, sgRNA backbone sequence and polymerase by PCR. A type III transcriptional terminator, which assembles multiple sgRNA expression cassettes into lentiviral plasmid 5 using the Golden Gate cloning method to obtain a lentiviral plasmid in which a plurality of sgRNAs are co-expressed.

In another aspect of the invention, a method of constructing a gene knockdown in a particular subset of neurons is provided. include:

(1) providing an initial lentiviral plasmid, the first lentiviral plasmid comprising a first human U6 promoter, a stuffer sequence, a second sgRNA backbone sequence, a polymerase type III transcription terminator, an EFS promoter, Nucleic acid sequence elements such as dCas9-KRAB, nuclear localization signals, P2A, EGFP, and WPRE.

(2) Restriction endonucleases EcoRI and XbaI digest the initial lentiviral plasmid 1 and remove the original EFS promoter.

(3) PCR amplification of a neuron sub-population-specific promoter, adding an initial lentiviral plasmid at the 5' end of the PCR primer - removing the homologous arms and EcoRI/XbaI restriction sites on both sides after removal of the EFS promoter, preferably The homology arm is 15-20 bp.

(4) Homologous recombination The amplified neuron subpopulation-specific promoter was cloned into the initial chronic virus plasmid after digestion with EcoRI/XbaI, replacing the original EFS promoter.

The invention is described below with reference to the specific embodiments, which are intended to be illustrative, and are not to be construed as limiting. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and can be referred to the third edition of the Molecular Cloning Experiment Guide or related products, and the reagents and products used are also available. Commercially obtained. The various processes and methods not described in detail are conventional methods well known in the art, the source of the reagents used, the trade name, and the necessity to list the components thereof, which are indicated on the first occurrence, and the same reagents used thereafter are not The descriptions are the same for the first time.

Example 1 Inhibition of Target Gene Expression by Primary Neurons Infected with CRISPRi Lentivirus

First, a single sgRNA/CRISPRi-integrated expression plasmid targeting the gene in the neuron was constructed (Fig. 1a), and the sgRNA sequence is shown in Table 1 below. The lentiviral plasmid was digested with type II restriction endonuclease BsmbI at 37 ° C for about 1 h, and a 13 kb fragment was recovered by electrophoresis. The sgRNA primer was dissolved in 10 μM without DNAase water, and the mixture was annealed in an equal volume for 5 min at room temperature. The digested lentiviral plasmid was mixed with the annealed sgRNA at a ratio of 1:4, and an equal volume of Solution I (Takara) was added thereto, and ligated at 16 ° C for 1 h. The Stbl3 competent strain was transformed and sequenced. The endotoxin-free sgRNA/CRISPRi integration plasmid was purified, and then HEK 293FT cells were transfected with PEI, and lentiviruses were packaged with pVSVG, psPAX2 and sgRNA/CRISPRi (1:1.5:2). At the same time, we also selected the shRNA/RNAi of the targeted gene as a reference and purchased it directly from the TRC library. The hippocampus or cortical tissue of the nascent suckling mice were then dissected, trypsinized, pipetted and plated in a single cell in a polylysine coated 24-well plate. On the third day after primary neuronal culture, lentivirus was added at a multiplicity of infection (MOI) of 10, and on day 14 with RIPA lysate (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5). The cells were lysed with %sodium deoxycholate and 0.1% SDS and a mixture of protease inhibitors, and the cell lysate supernatant was collected by centrifugation at 12000 x g for 15 min, and then immunoblot analysis of the expression of the protein encoded by the target gene (Figs. 3b and 3c) or extraction of total RNA by Trizol (Fig. 3b and 3c) Life Technologies), Reverse Transcript cDNA (Life Technologies), qRT-PCR analysis of mRNA abundance of targeted gene transcription using 2X SYBR Green master mix (Bio-Rad) and gene-specific primers (see Table 2) (Figure 3d) ). The results showed that CRISPRi can efficiently inhibit the expression of targeted genes, both from mRNA and protein expression levels, with inhibition levels as high as 90%.

The highly efficient silencing of gene-expressed sgRNA in Example 1 was selected and the lentivirus was packaged. Lentivirus was added on the third day after primary neuron culture, and electrophysiological recording of action potential-induced EPSC in primary neurons infected with CRISPRi lentivirus and changes in expression of corresponding exogenous gene rescued phenotypes on the fifteenth to seventeenth days (Figures 3e and 3f). Because the four genes selected are involved in neurotransmitter release, their functional inactivation will inhibit the release of neurotransmitters. The results indicate that CRISPRi can significantly reduce the amplitude of the targeted gene EPSC. Thus, CRISPRi can effectively achieve the complete inactivation of the function of the targeted gene. At the same time, the exogenous gene rescue experiments also confirmed the specificity of CRISPRi targeting gene inactivation.

Table 1: sgRNA sequences and shRNA sequences that target genes in neurons.

Table 2: qRT-PCR primer sequences for targeting genes in neurons.

Example 2 assesses the specificity of the CRISPRi system for inhibiting targeted gene expression in primary neurons

Time-curve analysis of CRISPRi inhibition of gene expression. Syt1sgRNA2 and Syt1 shRNA1, which efficiently silence Syt1 expression in Example 1, were selected, packaged with lentivirus and infected with primary neurons cultured for three days, and then lysed with RIPA on days 5, 6, 8, 10, 12 and 14 respectively. Cell lysates were collected and analyzed by immunoblotting for the expression of Syt1 over time (Figs. 4a and 4b). The results showed that the primary neurons were in the eighth day of culture, and the CRISPRi system began to exert the function of suppressing genes, and the inhibition level gradually increased with time. Although RNAi systems have similar time inhibition curves, their level of inhibition is always lower than that of the CRISPRi system.

Kinetic analysis of CRISPRi targeting genes. The Syt1 sgRNA2 of Example 1 was selected, the lentivirus was packaged and the primary cortical neurons cultured for three days were infected, and then the cells were collected on the fifth, sixth, eighth, tenth, thirteenth and fourteenth days, respectively. That is, the cells were first cross-linked with 1% formaldehyde for 10 minutes, and 125 mM glycine was terminated for 5 minutes, and the cross-linked cells were collected. The cells were then lysed with 1% SDS and the chromatin DNA was cleaved into fragments with a major peak at around 200-300 bp using a sonicator. The Syt1 sgRNA2/dCas9-KRAB targeting fragment was then enriched overnight by incubation with FLAG antibody at 4 ° C, and the enriched DNA was purified by cross-linking at 65 °C. Finally, ChIP-qPCR analysis of the binding of the CRISPRi system to the Syt1 gene over time (Fig. 4c). The results indicate that the binding site of the CRISPRi system has an increasing binding capacity over time, and reached half of the maximum binding threshold around the eighth day. Thus, in combination with the experimental results of the time curve analysis of CRISPRi inhibition of gene expression, we hypothesized that the CRISPRi system needs to achieve at least a half-maximal binding threshold for its inhibitory function.

The effect of sgRNA/DNA mismatch on CRISPRi inhibition of gene expression. A series of Syt1 sgRNA2 mutants (Fig. 4d) were designed, packaged with lentivirus and infected with primary cortical neurons cultured for three days. On the fourteenth day, 1% formaldehyde cross-linked cell samples were collected, FLAG antibody was enriched for the targeting fragment of Syt1 sgRNA2 mutant, and ChIP-qPCR was used to analyze the effect of sgRNA/DNA mismatch on the binding ability of Syt1 target site (Fig. 4e), and qRT. - PCR analysis (Fig. 4f) and immunoblot analysis (Fig. 4g) mismatched the effect of Syt1 gene expression. The results show that CRISPRi has a very low tolerance to sgRNA/DNA mismatch. When mismatched, dCas9-KRAB is difficult to target gene loci with high affinity, and thus does not inhibit gene expression and produce off-target effects.

The CRISPRi system inhibits the specificity of targeted gene expression in primary neurons. The sgRNA of the DsRed gene (see Table 1) was designed and cloned into a single sgRNA/CRISPRi-integrated expression plasmid. Primary neurons of three and five days after culture were infected with LsRed sgRNA/dCas9-KRAB and exogenous Syn::DsRed, respectively. On the fourteenth day, Trizol extracted total RNA, which was then sent to the Huada Gene Bank for RNA-seq transcription sequencing analysis. The results showed that sgRNA targeting DsRed significantly inhibited the mRNA abundance of DsRed, but did not significantly change the expression of other genes. Thus the CRISPRi system has excellent targeting gene specificity in primary neurons (Fig. 4h).

Example 3 uses CRISPRi to achieve selective inactivation of genes in specific neuronal subpopulations

This example uses a promoter specific for the expression of specific neuronal subtypes to drive dCas9-KRAB expression in specific neuronal types, but not in other types of neuronal subpopulations, to achieve targeted gene selectivity in a given subset of neurons. Inactivated. Selecting the highly efficient silencing gene-expressing Syt1 sgRNA2 in Example 1, and then replacing the vector EFS promoter with a neuronal subtype-specific mouse CamKIIα (excitatory neuron) or VGAT (GABA-inhibitory neuron) promoter (Figure 5a). The mouse CamKIIα and VGAT promoters were amplified with PCR primers (Table 3), and then the recombinant Syt1 sgRNA2 vector was digested with one step recombinant clone (Vazyme Biotech) to EcoRI/XbaI to replace the original EFS promoter.

Table 3: Neuronal subtype specific gene expression promoter PCR primer sequences.

1 μl of lentivirus (1.0×10 ¹⁰ infectious units/ml) stereolocalization (Bregma) targeting the excitatory and GABAergic inhibitory neuronal subpopulations of the above plasmids was injected into 4 week old mice (relative to the mouse) Bulk injection parameters: front and rear -2.0mm, left and right ±1.5mm and depth -2.5mm). Downstream analysis was performed two weeks later.

The mice injected with the virus were perfused with a 4% paraformaldehyde heart, and the brain was sliced into a 50 μm thick brain slice using a cryostat (Leica, VT1000S). 5% normal goat serum was blocked and anti-GFP or Syt1 antibodies were incubated with the corresponding fluorescently labeled secondary antibodies. The results showed that the lentivirus was able to infect granulosa cells in the dentate gyrus of the hippocampus of approximately 20% of the mice (Fig. 5b left). Moreover, CRISPRi is capable of specifically inducing excitatory and expression of Syt1 in a subgroup of GABAergic inhibitory neurons (Fig. 5b right).

Live cell sorting identified specificity of targeted cell subpopulations as well as selective inactivation of targeted genes (Fig. 5c). First use a live perfusate (115 mM choline-chloride, 2.5 mM KCl, 1.25 mM NaH ₂ PO ₄ , 26 mM NaHCO ₃ , 10 mM D-(+)-glucose, 8 mM MgSO ₄ , 1 mM L-ascorbate-Na, 3 mM Na-pyruvate ; pH 7.2-7.4) The mice injected with the virus were perfused and the brains of the mice were cut into brain slices using a brain piece mold, and then transferred to binary gas (95% O ₂ and 5% CO ₂ ) saturated EBSS buffer ( 116 mM NaCl, 5.4 mM KCl, 26 mM NaHCO ₃ , 1 mM NaH ₂ PO ₄ , 1.5 mM CaCl ₂ , 1 mM MgSO ₄ , 0.5 mM EDTA, 25 mM glucose, 1 mM cysteine), followed by dissection of the mouse hippocampus, further containing 20 units/ The EBSS buffer of ml Papain (Sigma) and 0.005% DNase I was digested at 37 ° C for 1 hour, and the binary gas was kept intact. Finally, the reaction was terminated with EBSS buffer containing 1 mg/ml ovomucoid, 1 mg/ml BSA and 0.005% DNase I. The supernatant was removed by centrifugation, and the tissue block was blown into a single cell suspension, and the cells were flowed with DAPI/EGFP. Sorting. The RNA in the living cells after sorting was amplified using the Smart-seq2 ²² technique.

qRT-PCR was used to detect the expression of Vglut1 and Gad1 in flow-sorted pCaMKIIα::dCas9-KRAB and pVGAT::dCas9-KRAB infected neurons, respectively (Fig. 5d), and the qRT-PCR primer sequences are shown in Table 2. The results showed that pCaMKIIα::dCas9-KRAB positive cells mainly expressed excitatory Vglut1, while pVGAT::dCas9-KRAB mainly expressed inhibitory Gad1.

qRT-PCR was used to detect the expression of Syt1 in flow-sorted pCaMKIIα::dCas9-KRAB and pVGAT::dCas9-KRAB infected neurons (Fig. 5e). The results showed that in the pCaMKIIα::dCas9-KRAB and pVGAT::dCas9-KRAB positive cells, Syt1 expression was inhibited only in the presence of Syt1 sgRNA2, whereas the control Scr sgRNA could not change Syt1 expression.

Western blot analysis of the expression of Syt1 in flow-sorted pCaMKIIα::dCas9-KRAB and pVGAT::dCas9-KRAB infected neurons (Fig. 5f). The results showed that in the pCaMKIIα::dCas9-KRAB and pVGAT::dCas9-KRAB positive cells, Syt1 expression was inhibited only in the presence of Syt1 sgRNA2, whereas the control Scr sgRNA could not change Syt1 expression. Moreover, there was no significant change in Syt1 expression in pCaMKIIα::dCas9-KRAB and pVGAT::dCas9-KRAB negative cells.

The highly efficient silencing of gene-expressed sgRNA in Example 1 was selected and the lentivirus was packaged. Lentivirus was added on the third day after primary neuron culture, and electrophysiological recording of action potential-induced EPSC in primary neurons infected with CRISPRi lentivirus and changes in expression of corresponding exogenous gene rescued phenotypes on the fifteenth to seventeenth days (Figures 5g and 5h). The results showed that in pCaMKIIα::dCas9-KRAB positive cells, Syt1 sgRNA2 attenuated EPSC amplitude relative to Scr sgRNA, and was able to rescue phenotypic changes by exogenous Syt1; IPC amplitude was not affected in pCaMKIIα::dCas9-KRAB negative cells influences. On the other hand, in pVGAT::dCas9-KRAB positive cells, Syt1 sgRNA2 attenuated IPSC amplitude relative to Scr sgRNA, and was able to rescue phenotypic changes by exogenous Syt1; EPSC amplitude in pVGAT::dCas9-KRAB negative cells Not affected. Since the entire network is composed of excitatory and inhibitory cells, the selective inactivation of Syt1 in excitatory and inhibitory cells will disrupt the excitation/suppression balance of the entire neural network. In summary, the viral delivery CRISPRi system can be used to rapidly establish neuronal and animal models of neurobiology research.

Example 4 Assessment of Biological Function Changes in Animal Models Based on CRISPRi

The water maze test measures the learning ability of mice (Figures 6a and 6b). Water was injected into a circular tank of 1.2 m in diameter (water temperature 24 ° C, 30 cm deep), the shape of the pool was marked with different color shapes as reference road signs, and the 6 cm diameter escape platform was placed in a fixed quadrant. In the training phase, the mice were placed in the pool for 1 min to freely search for the escape platform, and let it stay on the platform for 10 s. If not found, put it directly on the platform for 10 s. Mice were trained 4 times a day for 40 consecutive days. In the test phase, the escape platform was removed on day 9 and the mice were placed in the pool for 1 min. The mice were recorded in the escape platform area and the dwell time in each quadrant. The results indicated that in the training phase, Syt1sgRNA2/pCaMKIIα::dCas9-KRAB mice (hereinafter referred to as pCaMKIIα mice) took longer to find the escape platform in each day of training compared to the control mice; and Syt1 sgRNA2/pVGAT The ::dCas9-KRAB mouse (hereinafter referred to as pVGAT mouse) can find the escape platform in a short time with respect to the control mouse in each day of training. In the test phase, pCaMKIIα mice stayed shorter in the plateau region and their quadrants, while pVGAT mice significantly prolonged their residence time in these regions. In conclusion, pCaMKIIα mice have poor learning and memory ability, while pVGAT mice have excellent learning and memory ability.

The Barnes labyrinth experiment tested mice's learning ability (Fig. 6c). The mice were starved to achieve 85% of their normal body weight. The mice were first placed in the Barnes Maze for 15 min for 2 consecutive days and the mice were rewarded with 0.2 g of chocolate. Each mouse then received 10 food search sessions and then 5 food tests. In the new hole test, mice were placed from the unmarked hole base (180° reversed from the Barnes Maze at the training stage) and tested 5 times per mouse. Record the time it takes for the mouse to find the food and place it in the storage hole as well as the path. The experimental results indicate that pCaMKIIα mice spend more time and travel farther into the storage hole in the original and new hole tests. In contrast, pVGAT mice are able to find shorter distances and take food for a short time to the storage hole.

The T maze test measures the learning ability of mice (Fig. 6d). The mice were starved to achieve 85% of their normal body weight. The mice were placed in the T-maze for 2 days in a familiar environment, 5 min/5 times per day, and the mice were rewarded with 0.2 g of chocolate. Then, the mice were trained for one continuous forced selection and one free choice for 8 consecutive days. The interval between the two selections was 20 s, and the training was performed 4 times a day. On the 9th day of the test, the interval between the two selections was 1 min, and a total of 4 tests were performed. Record the percentage of mice that are correctly alternating. The results show that pVGAT mice can make better and better choices, that is, have excellent learning and memory ability.

Fear memory was used to test the memory capacity of mice (Fig. 6e). During the training phase, the mice were placed in a conditional fear box for 6 min, then subjected to two consecutive sound stimuli (2 800 Hz, 75 dB, 20 s) followed by a foot electrical stimulation (0.7 mA, 2 s) at 3 min intervals. On the first day of the test, each mouse was returned to the conditional fear box for 6 minutes without any sound stimulation. On the second day of the test, each mouse was returned to the new conditional fear box for 6 minutes of activity, followed by two 20s of sound stimulation at intervals of 3 minutes. A stationary reaction is a stagnation reaction and is expressed as a percentage of the total test time. The results showed that compared with the control mice, pCaMKIIα mice showed less stagnation in association and auditory suggestive conditional fear, that is, learning and memory ability was less frequent; while pVGAT mice showed more prominent learning and memory ability.

The open field experiment tested the motor's athletic ability and anxiety-like emotions (Fig. 6f). Mice were placed in the open field (50 cm X 50 cm X 40 cm) for free for 10 min to measure the exercise capacity of the mice. The results show that both pCaMKIIα and pVGAT have a reduced residence time in the central region of the open field.

The elevated plus maze tested the anxiety-like emotions of the mice (Fig. 6g). The mice were placed with their heads facing the elevated cross maze open arms and facing away from the experimenter, placed at the junction of the open and closed arms, and allowed to move freely for 5 min in the maze. The total time the mice stayed in the open arms was recorded. The results showed that both pCaMKIIα and pVGAT had reduced residence time in the open arms of the elevated plus maze, and they had anxiety-like behavior.

The tail suspension experiment tested the depression-like emotions of the mice (Fig. 6h). The tail of the mouse was suspended with a tape for 6 min, and the time when the mouse gave up the struggle and remained stationary was recorded. The results showed that pCaMKIIα and pVGAT mice increased in resting time in the tail suspension experiment, and they had depression-like behavior.

The forced swimming test was used to test the depression-like emotions of the mice (Fig. 6i). The mice were placed in a cylindrical water bath for 6 min and the water temperature was maintained at 23-25 °C. The mice were stopped from struggling in the water and only the head was exposed to the surface of the water. The results showed that pCaMKIIα and pVGAT mice increased in resting time during forced swimming experiments, and they had depression-like behavior.

Example 5 Flexible and diverse CRISPRi multiplex gene knockdown strategy in mouse brain

Two sgRNA/CRISPRi-integrated expression plasmids targeting genes in neurons (Fig. 1b and Fig. 7a) were constructed, i.e., the tandem human U6 promoter and the human H1 promoter independently drive the respective sgRNA expression. First, the template plasmid 2 was amplified using the high-fidelity enzyme PrimeSTAR (Takara), and the amplification primer sequences are shown in Table 4 below. Then, the PCR amplified fragment and the lentiviral plasmid were digested with a type II restriction endonuclease BsmbI at 37 ° C for about 1 h, and recovered by electrophoresis. Finally, add Solution I (Takara) and connect for 1 h at 16 °C. The Stbl3 competent strain was transformed and sequenced. An integrated CRISPRi expression plasmid with two sgRNAs inserted was obtained.

Construction of three sgRNA/CRISPRi-integrated expression plasmids targeting genes in neurons (Fig. 1c and Fig. 7a), independently driven by tandem human U6 promoter, human 7SK promoter and murine U6 promoter Individual sgRNA expression. Template plasmid III was first digested with type II restriction endonuclease BbsI at 37 °C. The second sgRNA primer was dissolved in 10 μM without DNAase water, and the mixture was annealed in an equal volume for 5 min at room temperature. The digested template plasmid 3 was mixed with the sgRNA at a ratio of 1:4, and an equal volume of Solution I (Takara) was added thereto, and ligated at 16 ° C for 1 h. The Stbl3 competent strain was transformed and sequenced to obtain template plasmid three inserted into the second sgRNA after the human 7SK promoter. The high-fidelity enzyme PrimeSTAR (Takara) then amplifies template plasmid three into which the second sgRNA has been inserted, and the amplification primer sequences are shown in Table 4 below. Then, the PCR amplification fragment and the lentivirus plasmid were digested with the type II restriction endonuclease BsmbI at 37 ° C for about 1 h, and electrophoresed. Finally, add Solution I (Takara) and connect for 1 h at 16 °C. The Stbl3 competent strain was transformed and sequenced. An integrated CRISPRi expression plasmid inserted into three sgRNAs was obtained.

Construction of five sgRNA co-expression plasmids targeting genes in neurons (Fig. 2a and Fig. 7a), ie, the human U6 promoter in tandem drives sgRNA expression independently, and between the two adjacent U6/sgRNA expression cassettes Increase the spacer sequence to reduce inter-transcriptional interference. A single sgRNA/CRISPRi-integrated expression plasmid targeting the genes in neurons was first constructed. Each individual sgRNA expression plasmid was then PCR amplified, the primer sequences were amplified as described in Table 4 below, and the PCR amplified fragment was digested with BsmbI. Finally, each sgRNA expression cassette was ligated into lentiviral plasmid 5 with T4 ligase (NEB) to obtain five lentiviral plasmids co-expressed with sgRNA, and this was ligated with dCas9-KRAB-expressed lentiviral plasmid to realize multiple genes. Knock down.

The CRISPRi lentivirus targeting the multiplex gene was packaged in HEK293FT cells (Fig. 7a). Lentivirus was added on the third day after primary neuronal culture, total RNA was extracted with Trizol on day 14, qRT-PCR was used to analyze mRNA abundance of targeted gene transcription (Fig. 7b), or cell lysis was collected using RIPA lysate. The supernatant was then immunoblotted to analyze the expression of the protein encoded by the targeted gene (Figures 7c and 7d). The results showed that in primary cultured neurons, both integrated vector and binary vector strategies can specifically inhibit the mRNA and protein expression levels of multiple targeted genes (2-5), inhibiting efficiency and targeting individual genes. The efficiency is quite.

At the same time, two different integrated CRISPR- or binary lentivirus mixtures targeting multiple genes were stereotactically injected into four-week-old mice. Two weeks later, live cell sorting identified inactivation of targeted multiple genes (Fig. 7e). The results showed that consistent with the primary cultured neurons, both the integrated vector and the binary vector could specifically inhibit the mRNA and protein expression levels of multiple targeted genes (2-5) in the living brain, and the inhibition efficiency. It is equivalent to the inhibition efficiency of targeting a single gene in primary neurons.

Table 4: sgRNA cloning primer sequences targeting multiple genes.

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms is not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, various embodiments or examples described in the specification, as well as features of various embodiments or examples, may be combined and combined.

Although the embodiments of the present invention have been shown and described, it is understood that the above-described embodiments are illustrative and are not to be construed as limiting the scope of the invention. The embodiments are subject to variations, modifications, substitutions and variations.

Claims (10)

1. Use of sgRNA/CRISPRi for genetic engineering in non-dividing cells.
2. The use according to claim 1, wherein the non-dividing cells are neuronal cells.

   Optionally, the neuronal cell is an excitatory glutamatergic neuron or an inhibitory interneuron,

   Optionally, the genetic modification is achieved by:

   Introducing an endotoxin-free sgRNA/CRISPRi expression plasmid with pVSVG, psPAX2 into a recipient cell to obtain a lentiviral vector carrying sgRNA/CRISPRi, which is a sgRNA/CRISPRi targeting a gene; and utilizing the lentivirus Infecting the neuronal cells;

   Optionally, the recipient cell is a HEK 293FT cell;

   Optionally, the introduction is PEI mediated.
3. The use according to claim 2, wherein the gene comprises at least one selected from the group consisting of Syt1, Vamp2, Stx1a, Snap25, Stx1b, Doc2a, Doc2b, and DsRed.
4. The use according to claim 3, wherein the sgRNA targets the transcription initiation region of the gene from -50 bp to 300 bp downstream;

   Optionally, the sgRNA has the nucleotide sequence set forth in SEQ ID NOS: 1-15.
5. The use according to claim 4, wherein the sgRNA/CRISPRi expression plasmid is obtained as follows:

   (1) Providing an initial lentiviral plasmid comprising a human U6 promoter, a stuffer sequence, a sgRNA backbone sequence, a polymerase type III transcription terminator, an EFS promoter, a dCas9-KRAB, a nucleus Localization signal, P2A, EGFP and WPRE nucleic acid sequence elements;

   (2) The annealed sgRNA was inserted into the initial lentiviral plasmid digested with BsmbI to obtain an integrated expression plasmid of a single sgRNA/CRISPRi.
6. The use according to claim 4, wherein the sgRNA/CRISPRi expression plasmid is obtained as follows:

   (1) providing an initial lentiviral plasmid comprising a human U6 promoter, a stuffer sequence, a second sgRNA backbone sequence, a polymerase type III transcription terminator, an EFS promoter, dCas9-KRAB, Nuclear localization signal, P2A, EGFP and WPRE nucleic acid sequence elements;

   (2) providing a template plasmid comprising a first sgRNA backbone sequence, a polymerase type III transcription terminator, and a human H1 promoter nucleic acid sequence element;

   (3) amplifying the template plasmid by PCR, wherein the 3' end of the PCR forward primer includes a 5' end sequence of the first sgRNA backbone sequence, and the 5' end of the PCR forward primer has a BsmbI recognition sequence and To insert a first sgRNA sequence, the 3' end of the PCR reverse primer comprises the 3' end sequence of the human H1 promoter sequence, and the 5' end of the PCR reverse primer has a reverse complement sequence to be inserted into the second sgRNA And BsmbI recognition sequence;

   Preferably, the PCR forward primer and reverse primer have the nucleotide sequence shown in SEQ ID NO: 16 or 17;

   (4) After digesting the PCR amplification product in (3) with BsmbI, ligating with the initial lentiviral plasmid to obtain two sgRNA/CRISPRi expression plasmids,

   Preferably, the polymerase type III transcription terminator and the human H1 promoter nucleic acid sequence element are tightly linked.
7. The use according to claim 4, wherein the sgRNA/CRISPRi expression plasmid is obtained as follows:

   (1) Providing an initial lentiviral plasmid comprising a human U6 promoter, a stuffer sequence, a third sgRNA backbone sequence, a polymerase type III transcription terminator, an EFS promoter, dCas9-KRAB, Nuclear localization signal, P2A, EGFP and WPRE nucleic acid sequence elements;

   (2) providing a template plasmid comprising a first sgRNA backbone sequence, a polymerase type III transcription terminator, a human 7SK promoter, a second sgRNA sequence insertion site, and the second sgRNA sequence insertion site comprises BbsI recognition sequence, second sgRNA backbone sequence, polymerase type III transcription terminator, murine U6 promoter;

   (3) inserting the annealed second sgRNA sequence into the BbsI-digested template plasmid to obtain a template plasmid containing the second sgRNA sequence;

   (4) the template plasmid containing the second sgRNA sequence obtained by PCR amplification; wherein the 3' end of the PCR forward primer is the 5' end sequence of the first sgRNA backbone sequence, and the PCR forward primer The 5' end has a BsmbI recognition sequence and a first sgRNA sequence, and the 3' end of the PCR reverse primer includes the 3' end of the murine U6 promoter, and the 5' end of the PCR reverse primer has a third sgRNA Identifying sequences to complementary sequences and BsmbI;

   Preferably, the PCR forward primer and reverse primer have the nucleotide sequence shown in SEQ ID NO: 20 or 21;

   (5) the PCR amplification product in (4) is digested with BsmbI, and ligated to the initial lentiviral plasmid to obtain three sgRNA/CRISPRi expression plasmids;

   Preferably, the polymerase type III transcriptional terminator is tightly linked to a human 7SK promoter and a murine U6 promoter.
8. The use according to claim 4, characterized in that the sgRNA/CRISPRi binary expression plasmid is obtained as follows:

   (1) providing a first initial lentiviral plasmid comprising a sequentially linked EFS promoter, dCas9-KRAB, nuclear localization signal, P2A, EGFP and WPRE nucleic acid sequence elements;

   (2) providing a second initial lentiviral plasmid, constructing a single sgRNA/CRISPRi expression plasmid according to the method defined in claim 5;

   (3) amplifying an sgRNA expression cassette by PCR, the sgRNA expression cassette comprising a spacer sequence, a human U6 promoter, an sgRNA sequence, an sgRNA backbone sequence, and a polymerase type III transcription terminator;

   Preferably, there is a spacer sequence between adjacent sgRNA expression cassettes;

   Preferably, the spacer sequence is a lentiviral cPPT element;

   Preferably, the PCR amplification forward primer and reverse primer have the nucleotide sequences shown in SEQ ID NOS: 22 to 31;

   (4) A plurality of sgRNA expression cassettes were assembled into the second initial lentiviral plasmid using the Golden Gate cloning method to obtain an sgRNA/CRISPRi binary expression plasmid.
9. A method of constructing a gene inactivation model, characterized in that genetic modification is performed in non-dividing cells using sgRNA/CRISPRi to obtain the gene inactivation model;

   Optionally, the sgRNA/CRISPRi is as defined in any one of claims 2 to 8,

   Optionally, the non-dividing cells are neuronal cells,

   Optionally, the neuronal cell is an excitatory glutamatergic neuron or an inhibitory interneuron,

   Preferably, the gene inactivation model is a cell model or an animal model.
10. A gene inactivation model characterized in that the gene inactivation model is a cell model obtained by the method of claim 9.

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