A method for safety evaluation of gene editing technology using transgenic mice

Abstract

The present invention discloses a method for safety evaluation of gene editing technology using transgenic mice. The transposon vector carrying the base editor element and PBase mRNA are injected into the fertilized eggs of C57BL / 6J mice, and then transplanted into the ICR pseudo-pregnant female mouse recipient. The F0 mice that successfully integrated the corresponding base editor element are self-fertilized to establish transgenic mouse parents. Then, the parents with the corresponding base editor element integrated are mated to obtain transgenic mice with BE3, YE1‑BE3‑FNLS, ABE7.10 and BE7.11 integrated. F148A The transgenic mouse lines of the editors were used to conduct a safety evaluation of the gene editing technology, ensuring the comprehensiveness, completeness, and accuracy of the evaluation.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and relates to the construction of transgenic mice and a method for systematically evaluating the safety of gene editing technology. Background Art

[0002] The CRISPR / Cas9 system uses Cas9 nuclease and single-stranded guide RNA (sgRNA) to achieve gene modification by generating double-strand breaks (DSBs) of genomic DNA, and has become a widely used genome editing technology. In recent years, various types of editing tools have been developed based on the CRISPR / Cas9 system, including single-base editors CBEs for mediating C to T single-base mutations and single-base editors ABEs for mediating A to G single-base mutations, which are developed by fusing mutant Cas9 nucleases (such as dCas9 that only has DNA binding ability and completely loses DNA cleavage function or Cas9 nickase that can only cleave a single strand of DNA) with cytosine deaminase or adenine deaminase.

[0003] Although CBEs and ABEs have been widely used, studies have found that CBEs can produce off-target effects at the DNA and RNA levels in in vitro cultured cells, mouse embryos, and rice, and ABEs can produce a large amount of RNA off-target in human HEK293T cell lines. Although high-fidelity CBEs including YE1-BE3-FNLS and YE1-BE4 were developed by introducing YE1 mutations in rAPOBEC1 through protein engineering technology, and ABEs with high efficiency and low off-target in cell lines, namely ABE7.10, were developed by introducing F148A and V82G mutations in TadA. F148A 、miniABEmax V82G , but the off-target effects of these single-base editors in adult applications are still unclear.

[0004] A study reported that CBE was used to perform gene therapy on mice with hereditary tyrosinemia type I (HT1) disease model, and the off-target effects of CBE treatment in vivo were analyzed after culturing some liver single cells isolated from treated mice and performing whole genome sequencing (WGS) on the cultured cell monoclones. Although the study did not detect off-target mutations, the study only tested a relatively small number of liver cells, which may have missed some edited cells that actually experienced off-target mutations. In other in vivo gene editing application studies, only sgRNA-dependent off-targets were detected, ignoring the potential off-target effects of sgRNA-independent deaminases in base editors (e.g., Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing. Nature biomedical engineering 5, 169-178, Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature 595, 295-302, In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice. Nature 589, 608-614). Therefore, the current in vivo off-target effect evaluation methods of single-base editors have certain limitations, and new detection methods are needed for systematic and comprehensive evaluation. In addition, single-base editors can be delivered to adults via different delivery tools. At present, adeno-associated virus AAV is widely used in adult delivery of gene editing tools due to its safety, low immunogenicity, and the advantages of being delivered to various tissues and cells. However, studies have reported that genes delivered by AAV can be continuously expressed in vivo for half a year or even several years. Therefore, the risks of long-term expression of these editing tools in adults also need to be comprehensively and systematically evaluated. Summary of the invention

[0005] The purpose of the present invention is to provide a method for evaluating the safety of gene editing technology using transgenic mice.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for evaluating the safety of gene editing technology, the method comprising the following steps:

[0008] A first transgenic animal is constructed, wherein the first transgenic animal contains a first vector for integrating and expressing a gene editing tool element into the genome of a first starting experimental animal (i.e., a wild-type animal) after being introduced into the animal; and the first transgenic animal is subjected to genomic mutation analysis, transcriptome mutation analysis, reproductive development monitoring, or phenotypic (e.g., tumors, obesity, metabolic abnormalities, etc.) monitoring.

[0009] Preferably, the first vector is integrated into the genome of a first starting experimental animal (eg, mouse, etc.).

[0010] Preferably, the gene editing tool is selected from cytosine base editors (CBEs) such as BE3, YE1-BE3-FNLS, and ABE7.10 F148A Any of the adenine base editors (ABEs) mentioned above.

[0011] Preferably, the evaluation method also includes the following steps: constructing a second transgenic animal, which contains a second vector for integrating the genome of a second starting experimental animal (i.e., a wild-type animal) and expressing a guide element for targeted editing of the gene editing tool after being introduced into the animal's genome; hybridizing the second transgenic animal with the first transgenic animal, and using the offspring obtained from the hybridization to analyze the off-target effects of the gene editing tool.

[0012] Preferably, the second vector is integrated into the genome of a second starting experimental animal (eg, mouse, etc.).

[0013] Preferably, the second vector is a sgRNA expression vector of the gene editing target gene.

[0014] A method for preparing an animal model, the method comprising the following steps:

[0015] A first transgenic animal is constructed, wherein the first transgenic animal contains a first vector for integrating and expressing a gene editing tool element into the genome of a first starting experimental animal (i.e., a wild-type animal) after being introduced into the animal; and the first transgenic animal is self-pollinated.

[0016] Preferably, the preparation method further comprises the following steps: allowing the offspring of the transgenic animals obtained by self-pollination to develop normally and monitoring weight changes during the development process until a third transgenic animal is obtained whose weight is significantly greater than the weight of the starting experimental animal at the corresponding developmental stage (i.e., obese).

[0017] Preferably, the method for constructing the first transgenic animal specifically includes the following steps: injecting the transfection system containing the gene editing tool expression vector into the fertilized egg of the first starting experimental animal, and then obtaining the F0 generation animal through embryonic development and production in the recipient animal; selfing the F0 animal to obtain the parents of the first transgenic animal, and mating the parents of the first transgenic animal to obtain the first transgenic animal. After replacing the expression vector, this construction method is also applicable to the construction of the second transgenic animal.

[0018] Preferably, the first starting experimental animal is a mouse, and the weight monitoring of the offspring of the transgenic animals obtained by self-breeding begins at birth and continues for more than 16 weeks (for example, until 30 weeks).

[0019] Preferably, the gene editing tool is BE3, and male offspring obtained by self-pollination are selected for weight monitoring.

[0020] Preferably, the gene editing tool is YE1-BE3-FNLS, and female offspring obtained by self-pollination are selected for weight monitoring.

[0021] The beneficial effects of the present invention are embodied in:

[0022] The present invention detects and analyzes the off-target effects of gene editing tools (such as single-base editors) by using transgenic mice that integrate gene editing tools (such as single-base editors) in vivo. Not only does it construct transgenic tool mice that can be used for disease modeling and other aspects; it also provides an evaluation system for the safety of gene editing tools used in vivo, ensuring the comprehensiveness, systematicness and accuracy of the evaluation results. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the process for constructing various strains of transgenic mice (A), the gene expression cassette used to construct transgenic mice (B), the birth rate of F0 mice and the base editor integration positive rate (C), and the expression of base editors in transgenic mice (D, E, F).

[0024] Figure 2 The process of transfecting transgenic mice with sgRNA expression vectors (A, D), target site base editing (B, E), and base editing efficiency (C, F).

[0025] Figure 3 The figures show the overall situation of SNVs induced by base editors on a genome-wide scale in vivo (A), statistics of SNV mutation types (B), genomic mutation analysis of the hybrid offspring between sgRNA transgenic mice targeting the coat color-related gene Tyr and transgenic mice integrated with different base editor elements (C, D, E), and off-target effect analysis of the genome of the hybrid offspring (F, G, H, I).

[0026] Figure 4 Statistics of the number of RNA SNVs induced by base editors in the transcriptome of different tissues in vivo (A), correlation analysis between RNA SNVs and base editor expression levels (B, C), and RNA off-target effect analysis (D, E, F, G).

[0027] Figure 5 Reproductive status of transgenic mice that have integrated base editor elements (A, B, C), weight monitoring of offspring of transgenic mice (D, E, F, G, H, I, J), and analysis of off-target effects of genes in adipose tissue (K, L). DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention, but not to limit the scope of protection of the present invention.

[0029] The present invention proposes a method for safety evaluation of gene editing tools by transgenic mice (Systematic evaluation Approach For gene Editing tools by Transgenic mIce, SAFETI). The method prepares a series of transgenic mouse strains that continuously express cytosine base editors (CBEs) BE3, YE1-BE3-FNLS and adenine base editors (ABEs) ABE7.10, hA3A-BE3, ABE7.10 F148A The side effects of long-term expression of these base editors in vivo were then systematically evaluated on a genome-wide and transcriptome-wide scale, as well as in phenotypes such as mouse reproduction and growth and development.

[0030] 1. Preparation of transgenic mice expressing base editors

[0031] In order to evaluate the effect of base editors in vivo, we first obtained the expression of BE3, YE1-BE3-FNLS, hA3A-BE3, ABE7.10, ABE7.10 by piggyBAC transposon integration. F148A The transgenic mouse strains were constructed as follows ( Figure 1 A):

[0032] 1) Introduce a CAG promoter, a base editor (BE3, YE1-BE3-FNLS, hA3A-BE3, ABE7.10 or ABE7.10) into the transposon vector backbone. F148A ) element, P2A linker peptide and enhanced green fluorescent protein eGFP gene expression cassette ( Figure 1B), the specific construction methods of these transposon vectors carrying base editor elements can be referred to the literature (Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver. Protein Cell 11, 518-524). At the same time, a transposon vector expressing only the eGFP gene was constructed as a control.

[0033] 2) The transposon vector and PBase mRNA were injected into C57BL / 6J mouse (wild type, WT) fertilized eggs (Zygote), and then transplanted into ICR pseudopregnant female mice recipients. The results showed that injection of BE3, YE1-BE3-FNLS or ABE7.10 F148A The transposon vector containing the element had no obvious harmful effect on the birth rate of F0 mice; and through fluorescence detection and PCR genotyping, it was found that more than 70% of the born pups had the corresponding base editor element inserted into the genome ( Figure 1 C. Figure 1 D), that is, the number of positive mice that successfully integrated the corresponding base editor elements was more than 70% of the total number of mice born. In contrast, the birth rate of pups injected with transposon vectors containing hA3A-BE3 or ABE7.10 elements was significantly reduced, and no positive mice were found by fluorescence detection and PCR genotyping ( Figure 1 C) The F0 mice that have successfully integrated the corresponding base editor elements were self-fertilized to establish transgenic mouse parents. The parents that have integrated the corresponding base editor elements were then mated to produce transgenic mice that have integrated BE3, YE1-BE3-FNLS, ABE7.10, and BE7.11. F148A A control group, i.e., a transgenic mouse line integrating the eGFP gene (referred to as GFP mice), was obtained in the same manner.

[0034] 3) In 8-week-old GFP mice and mice that had integrated BE3, YE1-BE3-FNLS, ABE7.10 F148A RNA-seq analysis was performed on the brain, lung, heart, liver, spleen, kidney, testis, ovary, muscle, and adipose tissues of transgenic mice with different base editor elements. The results showed that the expression of base editors in the muscle and heart tissues of transgenic mice with different base editor elements was significantly higher than that in other tissues, and the relative expression of YE1-BE3-FNLS in various tissues was higher than that of BE3, ABE7.10, and YE1-BE3-FNLS in various tissues. F148A ( Figure 1 E). Western blot analysis confirmed the protein expression of these base editors in different tissues, namely, they were highly expressed in heart and muscle tissues. Similarly, YE1-BE3-FNLS showed higher protein expression levels than the other two base editors in all tissues tested ( Figure 1 F), Figure 1 F also shows that there is no expression of the corresponding base editor in tissues of wild-type and GFP mice.

[0035] 2. Verification of gene editing function in transgenic mice expressing base editors

[0036] 2.1 Activity of C to T base editing in transgenic mouse cells incorporating BE3 and YE1-BE3-FNLS elements

[0037] Construct a U6-Hpd sgRNA vector (for specific construction methods, refer to Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver. Protein Cell 11, 518-524), which contains an sgRNA targeting the gene Hpd, and performs C to T editing at the target site recognized by the sgRNA ( Figure 2 B) Introduction of a stop codon into the Hpd gene in transgenic mice rescues the lethal phenotype of hereditary tyrosinemia type 1 in mice.

[0038] The vector was packaged into AAV8 virus and injected into the tail vein of 8-week-old transgenic mice that had integrated BE3 and YE1-BE3-FNLS elements. Two weeks after injection, hepatocytes were isolated by liver perfusion ( Figure 2 A). Sanger sequencing demonstrated that transgenic mice incorporating the BE3, YE1-BE3-FNLS elements had C to T editing at the target site ( Figure 2 B). High-throughput deep sequencing confirmed the C-to-T editing again. For example, at the C6 position (the sixth C base of the target site), the editing efficiencies of BE3 and YE1-BE3-FNLS were 20% and 30%, respectively ( Figure 2 C).

[0039] 2.2 Integrated ABE7.10 F148A A to G base editing activity in transgenic mice expressing the

[0040] Refer to Section 2.1 to construct the U6-DmdsgRNA vector targeting the gene Dmd (dysfunction of this gene causes Duchenne muscular dystrophy). This vector contains an sgRNA targeting the gene Dmd, which performs A to G editing at the target site recognized by the sgRNA ( Figure 2 E).

[0041] The U6-Dmd sgRNA vector was packaged into AAV9 virus and delivered to 8-week-old mice by local intramuscular injection. F148A The tibialis anterior muscle (TA muscle) of transgenic mice with the element was collected two weeks later. Figure 2 D) A to G edits were identified by Sanger and targeted deep sequencing, such as at position A6, ABE7.10 F148A The editing efficiency is 10.5% ( Figure 2 F).

[0042] 3.BE3, YE1-BE3-FNLS and ABE7.10 F148A Potential off-target effects in vivo

[0043] 3.1 Genomic DNA mutations in parental transgenic mice

[0044] DNA was extracted from muscle tissue (highly expressed corresponding genes) of 8-week-old transgenic mice that had integrated the corresponding base editor elements, and whole genome sequencing (WGS) was performed on the extracted DNA samples to analyze the DNA mutations induced by the base editors in vivo on a genome-wide scale. The results showed that 1,353 SNVs were found in the muscles of transgenic mice that had integrated the cytosine base editor BE3 element, which is more than five times the number of SNVs in GFP mice ( Figure 3 A), in which the number of C to T (or G to A) mutations was significantly higher than that of the GFP control group ( Figure 3 B) However, integration of YE1-BE3-FNLS, ABE7.10 F148A The number of SNVs in transgenic mice with the same element was not significantly increased compared with that in GFP mice ( Figure 3 A). The above results also indicate that BE3 can produce a large number of SNVs mutations in vivo.

[0045] 3.2 Genomic DNA mutations in offspring transgenic mice

[0046] Another transgenic mouse strain with Tyr sgRNA integrated was constructed by piggyBAC transposon integration (referred to as Tyr sgRNA mice: the transposon vector integrated into wild-type mice contains Figure 3C shows the CAG promoter, red fluorescent protein mCherry gene, U6 promoter and sgRNA targeting gene Tyr). After C to T editing of this gene site (Tyr), a stop codon will be introduced, causing the mouse coat to be albino. This strain of mice was crossed with transgenic mice that integrated base editor elements, and WGS sequencing analysis was performed on the tail DNA of the parents and the resulting offspring, and the on-target efficiency and de novo mutations of the offspring were analyzed. The mouse breeding combinations established in the experiment are as follows: GFP mice and transgenic mice that integrated BE3, YE1-BE3-FNLS, ABE7.10 F148A The transgenic mice with the BE3, YE1-BE3-FNLS, and ABE7.10 were used as the males and the Tyr sgRNA mice were used as the females. F148A The transgenic mice with BE3 and YE1-BE3-FNLS elements were used as the female parent, and the Tyr sgRNA mice were used as the male parent. The offspring's coat color was observed and it was found that the offspring obtained by hybridization with the Tyr sgRNA mice, which integrated the BE3 and YE1-BE3-FNLS elements as the parent (male or female), had black and white coat color ( Figure 3 D) WGS analysis of the offspring of transgenic mice with BE3, YE1-BE3-FNLS elements and Tyr sgRNA mice showed that the offspring had C to T editing at the target site and integrated ABE7.10 F148A The offspring of transgenic mice with the Tyr sgRNA produced A to G editing at the target site ( Figure 3 E). The above results demonstrate the single-base editing effect of these base editors at the target site in vivo.

[0047] By analyzing the newly generated mutations (de novo mutations) in newborn mice in addition to target site editing, the results showed that for the newly generated off-target effects in the offspring, whether the transgenic mice with integrated BE3 elements as fathers or mothers, their offspring had significantly more SNVs than the control group ( Figure 3 F. Figure 3 G). Further analysis of SNVs types revealed that the offspring of transgenic mice with integrated BE3 elements and Tyr sgRNA mice had significantly increased editing types from C to T and G to A ( Figure 3 H), and has a CBE conserved WCW (W = A or T) motif ( Figure 3 I), indicating that these newly generated SNVs were induced by the BE3 editor.

[0048] 4.BE3, YE1-BE3-FNLS and ABE7.10 F148AEffects of expression on RNA levels in different tissues in vivo

[0049] By detecting the transcriptome-wide RNA mutations and differential gene expression induced by base editors in the corresponding transgenic mouse strains and comparing them with GFP mice, it was found that the number of RNA SNVs in all tissues of transgenic mice with integrated BE3 elements increased, with a significant increase in 5 tissues, especially in muscle tissue, where the number of RNA SNVs in transgenic mice with integrated BE3 elements was the highest ( Figure 4 A). In addition, the number of induced RNA SNVs increased with the increase of BE3 expression ( Figure 4 B), these RNA SNVs mostly occur in genes with high expression in vivo ( Figure 4 C). Analysis of the proportion of different mutation types of muscle tissue RNA SNVs in different groups found that the proportion of C to T and G to A editing types in transgenic mice with integrated BE3 elements and transgenic mice with integrated YE1-BE3-FNLS elements reached 88% and 68%, respectively, which was higher than 24% in the control group ( Figure 4 D), and the RNA SNVs induced in transgenic mice that integrated BE3 and YE1-BE3-FNLS elements also had the conserved WCW motif of CBE ( Figure 4 E), indicating that these in vivo RNA SNVs are induced by BE3 or YE1-BE3-FNLS.

[0050] Cytosine base editors not only induce a large number of RNA mutations in the transcriptome in vivo, but also KEGG enrichment analysis of RNA SNVs in muscle tissue of transgenic mice with BE3 elements revealed that these mutations may affect insulin signaling pathways, lipid metabolism, and cancer signaling pathways ( Figure 4 F). In addition, BE3, YE1-BE3-FNLS, ABE7.10 were also found to be integrated F148A The transgenic mice with these elements had dozens or even thousands of genes differentially expressed in different tissues, indicating that these base editors can cause differential expression of different genes in the transcriptome in vivo ( Figure 4 G).

[0051] 5.BE3, YE1-BE3-FNLS and ABE7.10 F148A Effects on reproduction and body weight of transgenic mice

[0052] First, the fertility of transgenic mice with the corresponding base editor elements was investigated. Three pairs of transgenic mice were selected for mating from each transgenic mouse line. The mice were kept together for a total of 6 months, and the number of pups was counted. Although the litter size of transgenic mice with the BE3 element was slightly less than that of GFP mice ( Figure 5 A), but the total number of litters of each transgenic mouse line integrating the corresponding base editor element did not differ from that of GFP mice ( Figure 5 B), and the transgenic mouse line that integrated the base editor element produced pups with a similar sex ratio to that of GFP mice ( Figure 5 C) However, body weight monitoring at 66 weeks showed that the average body weight of the offspring of transgenic mice that integrated the BE3 element was significantly higher than that of the control group ( Figure 5 D). Specifically, there was no difference in birth weight between the offspring of transgenic mice with base editor elements and GFP mice, while the offspring of transgenic mice with BE3 elements gained more weight than the control group after 16 weeks, especially male mice. The corresponding male mice weighed (49.2±8.1g; n=31), which was significantly higher than the control group (37.5±4.4g; n=18). However, the offspring of transgenic mice with YE1-BE3-FNLS, ABE7.10 and BE3 elements gained more weight than the control group (49.2±8.1g; n=31). F148A The average body weight of the offspring of transgenic mice with the element was not significantly different from that of the control group ( Figure 5 D), it is worth noting that the average weight gain of female mice in the offspring of transgenic mice with the YE1-BE3-FNLS element integrated was also significantly higher than that in the control group starting from 30 weeks after birth ( Figure 5 D); Statistics show that more than half of the mice in each litter gained weight. The above results show that the extended expression of base editors has little effect on the reproductive capacity of transgenic mice, but has a significant effect on body weight.

[0053] To further determine the obesity phenotype of transgenic mice with BE3 elements integrated, the offspring of transgenic mice with BE3 elements integrated and GFP mice ( Figure 5 E), abdominal fat tissue (Abdominalfat) and liver (Liver) were taken, and it was found that the offspring of transgenic mice with integrated BE3 elements were significantly larger in body size ( Figure 5 F), and the corresponding abdominal adipose tissue and liver volume and weight were larger ( Figure 5 G. Figure 5 H); Histopathological analysis also showed that the fat cells in the adipose tissue of the offspring of transgenic mice with the BE3 element integrated were larger ( Figure 5 I), H&E staining also observed liver fat deposition and liver dysplasia in the offspring of transgenic mice that integrated the BE3 element ( Figure 5 J). In addition, WGS and RNA-seq were performed on the fat tissue of the offspring of transgenic mice with integrated BE3 elements and GFP mice at 30 weeks of age (to explore the potential mechanism of overweight in transgenic mice). The results showed that the number of mutations in the genome of the offspring of transgenic mice with integrated BE3 elements was significantly higher ( Figure 5 K). And these mutations mainly occur in metabolism-related pathways, insulin secretion-related pathways, adipocyte differentiation and lipid storage-related pathways, etc. ( Figure 5 These results suggest that the obesity phenotype of transgenic mice may be caused by genome-wide mutations induced by BE3.

Claims

1. A method for evaluating the safety of gene editing technology, characterized in that: The evaluation method includes the following steps: Constructing a first transgenic animal, wherein the first transgenic animal contains a first vector for integrating and expressing a gene editing tool element into the animal genome after being introduced into a first starting experimental animal; performing genomic mutation analysis, transcriptome mutation analysis, reproductive development monitoring, or phenotypic monitoring on the first transgenic animal; The method for constructing the first transgenic animal specifically comprises the following steps: injecting a transfection system containing a gene editing tool expression vector into a fertilized egg of a first starting experimental animal, and then performing embryonic development and production in a recipient animal to obtain an F0 generation animal; selfing the F0 animal to obtain the parents of the first transgenic animal, and mating the parents of the first transgenic animal to obtain the first transgenic animal; The gene editing tool is a base editor; The evaluation method further comprises the following steps: constructing a second transgenic animal, wherein the second transgenic animal contains a second vector for integrating into the animal genome and expressing the guide element for targeted editing of the gene editing tool after being introduced into the second starting experimental animal; hybridizing the second transgenic animal with the first transgenic animal, and using the hybridized offspring to analyze the off-target effects of the gene editing tool; The second vector is an sgRNA expression vector of the gene editing target gene.

2. A method for evaluating the safety of gene editing technology according to claim 1, characterized in that: The first vector is integrated into the genome of the first starting experimental animal.

3. A method for evaluating the safety of gene editing technology according to claim 1, characterized in that: The gene editing tool is selected from any one of a cytosine base editor and an adenine base editor.

4. A method for evaluating the safety of gene editing technology according to claim 1, characterized in that: The second vector is integrated into the genome of the second starting experimental animal.

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