An antibody non-functional allele mouse model and a construction method and application thereof

Abstract

The application discloses an antibody non-functional allele and an antibody non-functional allele system, and further discloses an antibody non-functional allele mouse model. The application further discloses application of the antibody non-functional allele and the system thereof in constructing the antibody non-functional allele mouse model, detecting deletion and insertion of the antibody gene, analyzing a mechanism of long CDR3 generation, analyzing a mechanism of deletion and insertion of the antibody gene, and constructing a mouse model for improving a deletion and insertion frequency of the antibody gene. The application further discloses application of the antibody non-functional allele mouse model in screening a broad-spectrum neutralizing antibody and the like. The application further discloses an analysis method of the deletion and insertion of the antibody gene, and the like. The application has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and biomedical technology, and particularly relates to an antibody nonfunctional allele mouse model, its construction method and application, as well as a detection method for antibody gene deletion and insertion. Background Technology

[0002] Gene deletions and insertions are the removal or insertion of single or multiple nucleotides in the genome. In coding sequences, assuming deletions and insertions of different sizes occur at the same frequency, two-thirds of somatic cell-acquired deletions and insertions may be harmful to the cell due to frameshift mutations. In non-coding regions, small gene insertions can also be carcinogenic by altering cis-elements, such as forming super-enhancers that persistently activate oncogenes. Defects in DNA damage repair or DNA replication errors are associated with increased gene deletions and insertions, as well as frameshift mutations, in cancer. In immunoglobulin variable region exons, small deletions and insertions (<50 bp) arising from antibody diversification are generally detrimental to B cells. However, small deletions and insertions frequently occur in rare broadly neutralizing antibodies (bnAbs) and play an important role in their function. For example, in-frame deletions and insertions occurring at the antigen contact site of a broadly neutralizing anti-HIV antibody enhance the binding affinity of the antibody to the HIV envelope trimer epitope. Therefore, while deletions and insertions are generally harmful, in-frame deletions and insertions that occur during antibody diversification are beneficial. To date, the mechanisms by which beneficial deletions and insertions occur during antibody diversification remain unknown.

[0003] The variable regions of antibodies or B cell receptors (BCRs) diversify through two main programmed DNA damage processes: V(D)J recombination and somatic hypermutation (SHM). During bone marrow B cell development, V(D)J recombination forms different V region exons through the assembly of variable (V), diversity (D), and joining (J) regions. This antigen-independent process produces tremendous antibody diversity, particularly at complementarity determining region III (CDR3), which consists of V, D, and J fragments. The joining of long D and J fragments is most likely to produce long CDR3, a common feature of many broad-spectrum neutralizing antibodies. Upon stimulation by foreign antigens, mature B cells undergo somatic hypermutation in the germinal center (GC). SHM introduces a high frequency of point mutations and a small number of small gene deletions and insertions at the CDRs of the heavy chain (IgH) and light chain (IgL or IgK) genes of the BCR. B cells with mutations that increase affinity for antigens will survive through positive selection, while B cells with mutations that decrease affinity for antigens or are detrimental to the BCR will be eliminated through negative selection. Therefore, deletions and insertions in broadly neutralizing antibodies are most likely to occur during SHM. Although not yet confirmed, insertions that occur during SHM may also contribute to the production of long CDR3 in broadly neutralizing antibodies.

[0004] Activation-induced cytidine deaminase (AID), a member of the AID / APOBEC cytidine deaminase family, can initiate high-frequency somatic mutations and class switch recombination (CSR). AID deaminates cytidine to uridine. Over the past two decades, genetically engineered mouse models have revealed that different DNA damage repair pathways are involved in repairing AID damage, ultimately leading to different SHM or CSR outcomes. These different mutational outcomes result from varying repair processes of uracil produced by AID deamination via error-prone base excision repair (BER) or mismatch repair (MMR) pathways. Previous studies, through mutational analysis of the 3′ region of rearranged V(D)J exons—specifically, the unselected JH intron region—have revealed that the BER pathway is primarily responsible for C:G transversion and conversion mutations, while the MMR pathway is primarily responsible for A:T nucleotide mutations. For AID-initiated CSRs, the double-strand break repair (DSBR) pathway, non-homologous endjoining (NHEJ) pathway, or alternative endjoining (A-EJ) pathway is activated, responsible for joining the two broken DNA ends at the switch (S) region. However, the mechanism by which uracil produced by AID deamination is converted into gene deletions and insertions remains unresolved because deletions and insertions were either undetectable or detected too infrequently in past studies to show significant differences.

[0005] Research into the mechanisms of gene deletions and insertions is often hampered by several factors. First, deletions and insertions occur extremely infrequently, requiring ultra-deep sequencing for detection. Second, PCR or sequencing processes can introduce artificially induced deletion and insertion events, which are often difficult to distinguish from real-world occurrences. Third, deletions and insertions have a high false-detection rate, especially in repetitive regions of gene sequences. Finally, a robust in vivo model system is lacking to capture real-world deletion and insertion events under physiological conditions. Summary of the Invention

[0006] This invention performed ultra-deep sequencing (480 million sequencing reads, 146 million GC B cells, and 101 mice) on antibody nonfunctional alleles from 15 gene-edited mouse models, identifying previously overlooked low-frequency insertion events and 1bp deletion and insertion events. Furthermore, this invention proposes that deletions and / or insertions of different sizes occur at different frequencies. Detrimental 1bp deletions and insertions are prevalent, while deletions and insertions of longer fragments that produce antibodies are rare. The invention further proposes differences in the mechanisms that generate 1bp and long fragment deletions and / or insertions, which explains their different frequencies. Finally, this invention utilizes an antibody nonfunctional allele system to generate mouse models that increase deletions and / or insertions, which facilitates the screening of broad-spectrum neutralizing antibodies.

[0007] This invention overcomes the difficulties in detecting deletions and insertions by utilizing an antibody nonfunctional allele system. Furthermore, different gene knockout mice were constructed within this model to elucidate the mechanisms of antibody gene deletion and insertion during SHM (Self-Syndrome Injection). The antibody nonfunctional allele mouse model proposed in this invention overcomes problems related to CDR3 diversity and the rarity of gene insertions, revealing that AID-mediated insertion of repetitive sequences contributes to the production of antibodies carrying long CDR3s. This research is of great significance for developing animal models for testing HIV vaccines and inducing the production of broad-spectrum neutralizing antibodies.

[0008] This invention proposes a nonfunctional antibody allele, which refers to a gene sequence B1-8(H)(V) inserted into the antibody heavy chain region. H The B1-8 sequence is characterized by a gene deletion at the 3' end of the exon. This deletion occurs at the splice donor site, disrupting the normal splicing of the B1-8 exon with the downstream constant region exon mRNA, thus preventing the production of a functional B cell receptor. This sequence V... H The B1-8 protein has a cleavage site at the 3' end that is 2 bp, 12 bp, 14 bp, or 47 bp missing, so it cannot encode a protein and is not subject to selection pressure.

[0009] The gene sequence of the nonfunctional antibody allele described in this invention is shown in SEQ ID NO. 1-4 below:

[0010] SEQ ID NO.1: IgH 12

[0011]

[0012] SEQ ID NO.2: IgH 2

[0013]

[0014] SEQ ID NO.3: IgH 14

[0015]

[0016] SEQ ID NO.4: IgH 47

[0017]

[0018] The gene sequence of the antibody nonfunctional allele described in this invention may also be a sequence having 50%, 60%, 70%, 80%, or 90% or more homology with the sequence shown in SEQ ID NO. 1-4, or other sequences that can achieve the purpose of this invention.

[0019] The present invention also proposes the application of the antibody nonfunctional allele in constructing antibody nonfunctional allele mouse models, detecting antibody gene deletion and insertion, analyzing the mechanism of long CDR3 generation, dissecting the mechanism of antibody gene deletion and insertion, and constructing mouse models that increase the frequency of antibody gene deletion and insertion.

[0020] This invention also proposes an antibody nonfunctional allele system comprising the aforementioned antibody nonfunctional allele. The system inserts a rearranged VDJ sequence into the heavy chain region of a mouse antibody gene. Using the CRISPR / Cas9 system, a small gene deletion is generated at the 3' end of this VDJ sequence, disrupting the normal splicing of the VDJ exon with the downstream constant region exon mRNA, thus preventing the production of a functional B-cell receptor. This invention also proposes applications of the system in constructing antibody nonfunctional allele mouse models, detecting the characteristics of antibody gene deletion and insertion under different sequence backgrounds, and analyzing the mechanism of broad-spectrum neutralizing antibody production.

[0021] This invention also innovatively proposes an antibody non-functional allele mouse model. This mouse model is characterized by including one functional allele and one non-functional allele as described above in the antibody heavy chain allele. The functional allele successfully expresses the B cell receptor, maintaining B cell survival. The non-functional allele does not encode a protein and is not subject to selection pressure. Both alleles are transcribed through the same promoter.

[0022] The present invention provides two types of antibody-nonfunctional allele mouse models: model IgH H / 12 and model IgH WT / 12 Both mouse strains have a nonfunctional allele with a 12bp deletion of the V gene at the 3' end splice site. H B1-8 have different functional alleles: in one mouse model, the functional allele is V. HB1-8, named IgH H / 12 The first mouse model is characterized by identical sequences for both the functional and non-functional alleles, allowing them to serve as controls. The second mouse model uses the antibody heavy chain gene from wild-type mice, named IgH. WT / 12 This mouse model is characterized by functional alleles capable of generating random B-cell receptors through V(D)J rearrangement. This invention utilizes the mouse model IgH... H / 12 This invention successfully identified previously overlooked low-frequency insertion events and 1bp deletion / insertion events, and proposes that deletions / insertions of different sizes occur at different frequencies. Harmful 1bp deletions / insertions are common, while deletions and insertions of longer fragments that can generate antibodies are rare. Furthermore, this invention also found that the insertion of directly adjacent repeat sequences during SHM can extend the original 11-amino acid CDR3 to 18 amino acids, resulting in a long CDR3, which is one of the important characteristics of broad-spectrum neutralizing antibodies and autoantibodies.

[0023] This invention also provides an antibody-nonfunctional allele mouse model of IgH. WT / 12 Based on this, 12 DNA damage repair factors were knocked out to obtain mouse models of DNA damage repair factor knockout carrying antibody-nonfunctional alleles, including but not limited to IgH. WT / 12 Fen1 + / - IgH WT / 12 Ung - / - IgH WT / 12 53bp1 - / - IgH WT / 12 Atm - / - IgH WT / 12 Xlf - / - IgH WT / 12 Pms2 - / - IgH WT / 12 Mlh1 - / - IgH WT / 12 Msh2 - / - IgH WT / 12 Polh - / - IgH WT / 12 Exol - / - IgH WT / 12 Apex2 - / - IgH WT / 12 Aicda - / - A double-gene knockout mouse model of IgH was also obtained. WT / 12 Ung - / - Msh2 - / -This invention uses these mouse models to elucidate the generation mechanisms of 1bp and long fragment deletions and insertions during SHM, explaining the reason for their different frequencies.

[0024] This invention also provides an antibody-nonfunctional allele mouse model of IgH. WT / 12 Based on this, an antibody-nonfunctional allele mouse model of IgH was constructed that specifically overexpresses the exonuclease Trex2 in mature B cells. WT / 12 R26 + / Trex2 Cd21 Cre This invention utilizes this mouse model to significantly increase the frequency of long fragment deletions in antibody nonfunctional alleles.

[0025] This invention also provides an antibody-nonfunctional allele mouse model of IgH. WT / 12 Based on this, an antibody-nonfunctional allele mouse model of IgH was constructed by specifically knocking out DNA polymerase Polβ in mature B cells. WT / 12 Polb f / f Cd21 Cre This invention utilizes this mouse model to significantly increase the insertion frequency of long fragments of antibody nonfunctional alleles.

[0026] This invention also proposes a method for constructing an antibody-nonfunctional allelic mouse model, which utilizes an sgRNA targeting the 3' end of exon B1-8(H) to disrupt the normal splicing of exon B1-8 with downstream constant region exon mRNA, thereby preventing the production of functional B cell receptors; the method includes the following steps: designing an sgRNA targeting the 3' end of exon B1-8(H), with the sequence 5'-GGTTGTAAGGACTCACCTG-3' (SEQ ID NO.5). IgH is then used. H / H Mouse sperm and IgH WT / WT IgH was obtained from mouse oocytes using in vitro fertilization. WT / H The fertilized egg was then injected with sgRNA / Cas9 using the CRISPR / Cas9 system. WT / H From the fertilized eggs, four chimeric mouse models, namely the antibody nonfunctional allele mouse models, were finally obtained. These were mice with 2bp, 12bp, 14bp, and 47bp gene deletions at the 3' end of B1-8, named IgH. WT / 2 IgH WT / 12 IgH WT / 14 IgH WT / 47These deletions all occur at the splice donor site, disrupting the normal splicing of exons B1-8 with downstream constant region exon mRNA, thus preventing the production of functional B cell receptors. In a specific implementation, this invention selected a mouse strain (IgH) carrying a 12bp gene deletion. WT / 12 As a mouse model of antibody nonfunctional alleles, a series of mouse models were constructed based on it.

[0027] The construction method further includes the step of: using an antibody-nonfunctional allele mouse model IgH WT / 12 Based on this, CAG-Loxp-Stop-Loxp-TREX2-IRES-GFP was inserted into the Rosa26 site of mice to construct a Trex2-overexpressing mouse, namely R26. + / Trex2 Mice; the R26 + / Trex2 Mice and Cd21 mice carrying nonfunctional antibody alleles Cre Mice mated to obtain IgH WT / 12 R26 + / Trex2 Cd21 Cre Antibody-nonfunctional allele mouse model. Expression of Cre in mature B cells can remove the Stop component, allowing Trex2 and GFP to be successfully expressed under the action of the CAG promoter.

[0028] The construction method further includes the step of: using an antibody-nonfunctional allele mouse model IgH WT / 12 Based on this, Loxp was inserted flanking exon 3 of the Polb gene to obtain Polb. f / f The mice are Polβ conditionally knocked out; the Polβ... f / f Mice and Cd21 mice carrying nonfunctional antibody alleles Cre Mice mated to obtain IgH WT / 12 Polb f / f Cd21 Cre Antibody-free allele mouse model. Cre expression in mature B cells can cause deletion of exon 3 of Polb, resulting in loss of Polβ protein function.

[0029] The present invention also proposes the use of antibody nonfunctional allele mouse models, including the ability to detect low-frequency deletion and insertion events, the ability to detect long CDR3 generated during SHM, and the antibody nonfunctional allele mouse models can be used to analyze the mechanism of antibody gene deletion and insertion, and / or to explore and construct mouse models that increase the frequency of antibody gene deletion and insertion.

[0030] The applications include a mouse model with mature B cells overexpressing Trex2 that increases the frequency of antibody gene deletions, and a mouse model with mature B cells conditionally knocking out Polβ that increases the frequency of antibody gene insertions. These mouse models can increase the frequency of gene deletions or insertions in the original BCR library, thus improving the likelihood of screening for broad-spectrum neutralizing antibodies with gene deletions and insertions.

[0031] This invention also proposes a method for detecting extremely low frequency insertion events, the method comprising the following steps: Immunizing 8-12 week old antibody-nonfunctional allele mice (i.e., the antibody-nonfunctional allele mouse model) by subcutaneous injection of 200 μl of sheep blood and 20 μl of 0.5 μg / μl adjuvant 2'3'-c-diAM(PS)2(Rp,Rp)VacciGrade(Invivogen) on days 0 and 6, respectively. A booster immunization is performed on day 6 post-immunization using the same method. Based on the antibody-nonfunctional allele mouse model, spleens are harvested from mice on day 9 post-immunization and first processed using a B cell purification kit. The Negative Selection B Cell Enrichment Kit (Stem Cell Technologies) was used to purify spleen B cells. Then, biotin-labeled antibodies CD43, CD11c, and IgD were added, and non-germinal center B cells (CD43) were removed using magnetic bead sorting. + CD11c + and IgD + ), ultimately yielding 10 6 Germ center B cells with a purity of over 80% were used. Genomic DNA was extracted from the germ center B cells, and PCR amplification was performed using primers specific to non-functional alleles. Sequencing libraries were constructed, and high-throughput sequencing was performed, with each PCR amplicon sequencing approximately 5 million sequences. This method can obtain gene insertion events with a frequency as low as one in a thousand. The primers used to construct the sequencing library are shown in Table 1 below.

[0032] Table 1

[0033]

[0034]

[0035] Note: The bases at positions 27 to 32 in SEQ ID NO. 12 can be any other bases; the bases at positions 27 to 32 in SEQ ID NO. 13 can be any other bases; the bases at positions 27 to 32 in SEQ ID NO. 14 can be any other bases; the bases at positions 27 to 32 in SEQ ID NO. 15 can be any other bases; the bases at positions 27 to 32 in SEQ ID NO. 16 can be any other bases; the bases at positions 27 to 32 in SEQ ID NO. 17 are different for each sample in the same sequencing library;

[0036] In SEQ ID NO.19, the bases at positions 25 to 30 can be any other bases. In different sequencing libraries, the bases at positions 25 to 30 in SEQ ID NO.19 are different.

[0037] This invention also proposes bioinformatics analysis methods / definition methods for antibody gene mutations, deletions, and insertions. Based on the antibody nonfunctional allele mouse model constructed according to this invention, and based on the aforementioned method for detecting extremely low frequency insertion events, the results obtained from high-throughput sequencing are compared with the Bowtie2 and B1-8 reference sequences. H B1-8 has a point mutation at codon 92 (TGT > TGC). Therefore, sequences without this mutation are removed because these sequences may have originated from mouse V. H This gene is derived from gene amplification. This invention defines mutations, deletions, and insertions in sequencing results. If a nucleotide in the sequence differs from the reference sequence, and the Illumina sequencing quality score of that nucleotide is greater than 20, then that nucleotide is considered a point mutation. If the sequencing result aligns with the reference sequence and contains a gap, and the Illumina sequencing quality score of the four consecutive nucleotides flanking the gap is greater than 20, then the gap is considered a gene deletion event. If the sequencing result aligns with the reference sequence and contains a redundant sequence, and no two consecutive nucleotides in the sequencing result have an Illumina sequencing quality score less than 20, then this redundant sequence is considered a gene insertion event.

[0038] Gene mutation analysis mainly includes the analysis of total mutation frequency, the analysis of mutation frequency per nucleotide, the analysis of point mutation distribution, and the analysis of mutation maps. Total mutation frequency refers to the percentage of sequences containing point mutations out of the total number of sequences obtained from sequencing. Mutation frequency per nucleotide refers to the percentage of sequences containing a nucleotide mutation at a specific site out of the total number of sequences containing that site. Point mutation distribution is calculated by summing the mutation frequencies of every 20 bases to obtain its distribution map. Mutation map analysis is calculated by calculating the percentage of times a particular base mutates into another base out of the total number of times that base is detected in sequences with at least one point mutation.

[0039] The gene deletion and insertion analysis method proposed in this invention can comprehensively and rapidly analyze the characteristics of antibody gene deletions and insertions from aspects such as occurrence frequency, fragment size, and distribution features. It mainly includes analysis of total deletion or insertion frequency, analysis of deletion or insertion distribution, analysis of deletion or insertion frequency at different fragment sizes, and analysis of 1bp deletion or insertion distribution. Total deletion or insertion frequency refers to the percentage of the total number of deletions or insertions out of the total number of sequences obtained from sequencing. Deletion or insertion frequency at different fragment sizes refers to the percentage of deletions or insertions of a specific length out of the total number of sequences obtained from sequencing. The deletion or insertion distribution is calculated as the percentage of deletion start and termination sites or insertion sites per 20 bases out of the total number of sequences obtained from sequencing. The 1bp deletion or insertion distribution is calculated as the percentage of deletions or insertions at each site out of the total number of sequences obtained from sequencing. Deletions or insertions at sites with a SD greater than the mean are considered to be caused by human factors such as PCR and are therefore removed.

[0040] This invention also proposes a method for calculating and evaluating the similarity score between the inserted fragment and adjacent sequence bases. Based on the bioinformatics analysis method for antibody gene deletion and insertion, the method involves selecting the sequences on the left and right sides of the inserted sequence, which have the same length as the inserted sequence, comparing the bases at the same positions one by one, counting the number of bases with the same type, dividing by the total number of inserted bases, and obtaining the similarity scores between the left and right side sequences and the inserted sequence. The maximum value of the two is the final similarity score of the inserted sequence.

[0041] The innovations and beneficial effects of this invention include: the detection and mechanism analysis of rare antibody gene deletions and insertions, the elucidation of the long CDR3 production mechanism, and the proposal of a potential mouse model for vaccine research and broad-spectrum neutralizing antibody screening. This invention overcomes the difficulties in detecting deletions and insertions by utilizing an antibody non-functional allele system, and further constructs different gene knockout mice within this model, elucidating the mechanism of antibody gene deletions and insertions during SHM (Self-Survival Model). The antibody non-functional allele mouse model proposed in this invention overcomes problems related to CDR3 diversity and the rarity of gene insertions, revealing that AID-mediated repetitive sequence insertion contributes to the production of antibodies carrying long CDR3. This invention constructs a mouse model capable of increasing antibody gene deletions or insertions, which can be used in conjunction with humanized antibody mouse models for vaccine development and the production of broad-spectrum neutralizing antibodies. Attached Figure Description

[0042] Figure 1 Construction of antibody-nonfunctional allele mice. (A) Flowchart for constructing antibody-nonfunctional allele mice. IgH... H / H Mouse sperm and IgH WT / WT IgH was obtained from mouse oocytes using in vitro fertilization. WT / H The fertilized egg was then injected with sgRNA / Cas9 using the CRISPR / Cas9 system. WT / H (B) Strategy for generating antibody-nonfunctional alleles. CRISPR / sgRNA targeted the antibody heavy chain knock-in functional allele (Pro)B1-8, resulting in mice with a 12bp gene deletion at the 3' end of B1-8, which occurred at the splice donor site. This allele utilized a new splice donor site to produce a truncated mRNA that could not produce a functional BCR, and was therefore called the antibody-nonfunctional allele (Pas). (C) Four antibody-nonfunctional allele mouse models were obtained according to the method in Figure (A). Gene deletions of different sizes were generated at the splice donor sites of the edited functional alleles, resulting in truncated mRNAs. (D) Splenic B cells (B220) of heterozygous and homozygous antibody-nonfunctional allele mice. + CD4 - (E) Flow cytometry analysis. (F) Purification strategy for mouse immunization and GC B cells. (G) Flow cytometry analysis of purified GC B cells (B220). + PNA + The purity of ).

[0043] Figure 21bp deletions and insertions are common in SHM, while potentially functional deletions and insertions are very rare. (A) Experimental design flowchart. (B) Mutation frequency of Pro-Ig and Pas-Ig. (C) Deletion frequency of Pro-Ig and Pas-Ig. (D) Insertion frequency of Pro-Ig and Pas-Ig. (EF) Percentage of 1bp deletions and other deletions (E) or insertions (F) on the antibody non-functional allele. (GH) Percentage of in-frame and frameshift deletions (G) or insertions (H) on the antibody non-functional allele, with or without 1bp. (IJ) Percentage of A:T and C:G nucleotides in 1bp deletions (I) or insertions (J) on the antibody non-functional allele. The base composition of the sequence itself is labeled below the figure. (KL) Frequency of 1bp deletions (K) or insertions (L) per nucleotide on the antibody non-functional allele. The type of nucleotide inserted at hotspot locations is marked in the figure. The data in Figures (B), (C), (D), (I), (J), (K), and (L) were obtained by calculating the mean ± standard error of five independent mouse experiments. The p-values ​​in Figures (B), (C), (D), (I), and (J) were calculated using a two-tailed paired t-test. ****p < 0.0001, **p < 0.01, *p < 0.05. (MN) Comparison of deletion and insertion frequencies on the antibody nonfunctional allele in AID-deficient (n = 3) and control (n = 6) mice. (O) Percentage of in-frame and frameshift deletions and insertions within different fragment sizes on the antibody nonfunctional allele.

[0044] Figure 3 Insertion of repetitive sequences starting with .SHM can increase the length of CDR3. (A) An example illustrating the calculation of similarity scores between the inserted fragment sequence and neighboring sequences. (B) The proportion of inserts longer than 1 bp in Pro-Ig and Pas-Ig within different similarity score ranges. Data were obtained by calculating the mean ± standard error of five independent mouse experiments. (C) A schematic diagram of microhomologous sequences of different lengths. (D) The proportion of microhomologous sequences of different sizes in long fragment inserts longer than 1 bp. (E) The frequency of 1 bp and long fragment inserts at different locations on the antibody nonfunctional allele. (FG) An example illustrating that a 21 bp insertion near CDR3 in the mouse model of this invention and the anti-HIV broad-spectrum neutralizing antibody VRC08 extended the length of CDR3. The bases in the dashed boxes represent the repetitive sequence inserts and the replicated regions.

[0045] Figure 4SHM and CSR in DNA damage repair factor knockout antibody-nonfunctional allele mice. (A) Comparison of B cell number (left), GC B cell percentage (middle), and total GCB cell number (right) in spleens of different DNA damage repair factor knockout mice carrying antibody-nonfunctional alleles. The control group consisted of wild-type mice carrying only antibody-nonfunctional alleles. (B) The left figure shows the mutation frequency of the A:T site, the switching mutation frequency of the C:G site, and the transversion mutation frequency of the antibody-nonfunctional allele in different mutant mice. The right figure shows the statistical analysis of the left figure, with the circle color representing the mean difference relative to the control. - / - Msh2 - / - Msh2 - / - Ung - / The C:G conversion mutation in mice was increased relative to the control, while the others were decreased or showed no significant change relative to the control. The size of the circle represents the p-value. (C) Frequency of point mutations at different positions on the antibody nonfunctional allele. (D) Mutation profile analysis on the antibody nonfunctional allele. (E) IgG1 after 4 days of treatment of primary spleen B cells with anti-CD40 antibody and IL4. + Cell proportions. Data in Figure (AE) were obtained by calculating the mean ± standard error of independent mouse experiments. p-values ​​in Figures (A), (B), and (E) were obtained by one-way ANOVA with Bonferroni correction. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

[0046] Figure 5 Mechanisms of 1bp and long-fragment gene deletions and insertions. (AB) Frequency of 1bp and long-fragment gene deletions (A) and insertions (B) in different mutant mice. The right figure is the statistical analysis of the left figure, with the circle color representing the mean difference relative to the control. Among them, Msh2 - / - Deletion of 1bp and greater than 1bp in mice and Polh - / - In mice, deletions larger than 1 bp were increased relative to the control, while other deletions were decreased or showed no significant change relative to the control. The size of the circle represents the p-value. (C, E) Frequency of AID overexpression fragment insertion in different DNA polymerase-deficient cell lines. (D, F) Frequency of AID overexpression fragment insertion at different positions on the antibody-nonfunctional allele in different DNA polymerase-deficient cell lines. (GH) The heatmap shows the frequency of deletions (G) or insertions (H) of different fragment sizes. The color represents the mean difference relative to the control, where Fen1 + / - Pms2 - / - Mlh1 - / - Msh2- / - ,Polh - / - In mice, the deletions were either increased or showed no significant change compared to the control, while in other mice, the deletions were either decreased or showed no significant change compared to the control. (Pms2) - / - The 2bp insertion was increased in mice compared to the control group, Pms2 - / - Mlh1 - / - Msh2 - / - In mice, the 3bp insertion was increased compared to the control, while the other insertions were decreased or showed no significant change compared to the control. p-values ​​were calculated using Wilcoxon assays; values ​​greater than 0.05 were removed and indicated by white boxes. (IJ) Frequency of 4bp deletion (I) or 3bp insertion (J) at different locations on the antibody nonfunctional allele in different mutant mice. (KL) Frequency of 3-18bp deletion (K) or insertion (L) within boxes in different mutant mice. Figures (A), (B), (K), and (L) data were obtained by calculating the mean ± standard error of independent mouse experiments. P-values ​​were obtained using one-way ANOVA with Bonferroni correction: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Figures (C) and (E) data were obtained by calculating the mean ± standard error of independent cell experiments. Control CH12 cells (n = 4), Polb - / - (n=4), Poll - / - (n=4). The p-values ​​in Figures (C) and (E) were calculated using a two-tailed unpaired t-test. **p<0.01, *p<0.05.

[0047] Figure 6A mouse model capable of increasing gene deletion and insertion. (AB) Overall deletion frequency (A) and deletion frequency (B) of different fragment sizes on the antibody nonfunctional allele in control mice and Trex2 overexpressing mice. (C) Frequency of long fragment deletions at different positions on the antibody nonfunctional allele in control and experimental mice. (DE) Overall insertion frequency (D) and insertion frequency (E) of different fragment sizes on the antibody nonfunctional allele in control mice and Polβ conditional knockout mice. (F) Frequency of long fragment insertions at different positions on the antibody nonfunctional allele in control and experimental mice. The data in Figure (AF) are obtained by calculating the mean ± standard error of independent mouse experiments. Figure (AC) shows control mice (n=5) and Trex2 overexpressing mice (n=3). Figures (D-F) show control mice (n=3) and Polβ conditional knockout mice (n=3). The p-values ​​in Figures (A), (B), (D), and (E) were calculated using a two-tailed unpaired t-test. ***p < 0.001, **p < 0.01. (G) The radar chart summarizes the mutation profiles, CSR levels, and deletion and insertion frequencies of different fragment lengths in 15 different mouse genotypes.

[0048] Figure 7 Mechanistic models of antibody gene deletion and insertion. (A) Mechanism of AID initiation gene deletion and insertion. (B) Generation of 1bp and long fragment gene deletions and insertions depends on different factors / pathways. Detailed Implementation

[0049] The invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the invention are all common knowledge and general knowledge in the art, and the invention does not have any particular limitations.

[0050] Example 1. Using antibody nonfunctional allele mice, the low-probability event of antibody gene deletion and / or insertion can be observed.

[0051] To elucidate the characteristics and mechanisms of gene deletions and / or insertions, this invention constructs a novel antibody-nonfunctional allele mouse model, and based on this, constructs mouse models with knockout of 12 DNA damage repair factors. These include IgH... WT / 12 Fen1 + / - IgH WT / 12 Ung - / - IgH WT / 12 53bp1 - / - IgH WT / 12 Atm - / - IgH WT / 12Xlf - / - IgH WT / 12 Pms2 - / - IgH WT / 12 Mlh1 - / - IgH WT / 12 Msh2 - / - IgH WT / 12 Polh - / - IgH WT / 12 Exo1 - / - IgH WT / 12 Apex2 - / - IgH WT / 12 Aicda - / - In addition, a double-gene knockout mouse model of IgH was also obtained. WT / 12 Ung - / - Msh2 - / - .

[0052] First, the present invention is in V n B1-8 gene knock-in mouse zygotes were used to induce V... H A gene deletion occurred at the normal splicing site of B1-8, disrupting the normal splicing of exon mRNA in region V. Figure 1 Flow cytometry results showed that there were no B cells in the spleen of homozygous mice carrying the gene deletion, indicating that this allele produced nonfunctional BCRs. Figure 1 D). This invention selected mice carrying a 12bp gene deletion (IgH). 12 As a mouse model of antibody nonfunctional alleles, a series of mouse models were created based on it.

[0053] To detect the low-probability events of antibody gene deletion and insertion, this invention makes several improvements to the experimental method. First, an optimized immunization protocol and GC-B cell purification method are used to increase the number of GC-B cells to approximately 1 million for sequencing sample preparation. Second, the sequencing depth of each PCR amplicon is increased to approximately 5 million sequences, about 2500 times greater than previous studies. Simultaneously, this invention also constructs a bioinformatics program for analyzing antibody gene deletion and insertion, which can rapidly analyze the frequency, fragment size, and distribution characteristics of antibody gene deletions and insertions. Figure 1 EF, Figure 2A) Specifically, if a nucleotide in the sequence differs from the reference sequence and its Illumina sequencing quality score is greater than 20, then the nucleotide is considered to have undergone a point mutation. If the sequencing result aligns with the reference sequence and contains a gap, and the Illumina sequencing quality scores of the four consecutive nucleotides flanking the gap are greater than 20, then the gap is considered a gene deletion event. If the sequencing result aligns with the reference sequence and contains a redundant sequence, and no two consecutive nucleotides in the sequence have an Illumina sequencing quality score less than 20, then this redundant sequence is considered a gene insertion event. Gene mutation analysis mainly includes the analysis of total mutation frequency, the analysis of mutation frequency per nucleotide, the analysis of point mutation distribution, and the analysis of mutation maps. Total mutation frequency refers to the percentage of sequences containing point mutations out of the total number of sequences obtained from sequencing. The mutation frequency of each nucleotide refers to the percentage of sequences containing a nucleotide mutation at a specific site out of the total number of sequences containing that site. The point mutation distribution is calculated by summing the mutation frequencies of every 20 bases to obtain its distribution map. The mutation map is calculated as the percentage of times a particular base is mutated into another base out of the total number of times that base is detected in sequences with at least one point mutation. Gene deletion and insertion analysis mainly includes the analysis of total deletion or insertion frequency, deletion or insertion distribution, deletion or insertion frequency of different fragment sizes, and 1bp deletion or insertion distribution. Total deletion or insertion frequency refers to the percentage of the total number of deletions or insertions out of the total number of sequences obtained from sequencing. Deletion or insertion frequency of different fragment sizes refers to the percentage of deletions or insertions of a specific length out of the total number of sequences obtained from sequencing. The deletion or insertion distribution is calculated as the percentage of the number of deletion start and stop sites or insertion sites per 20 bases out of the total number of sequences obtained from sequencing. The 1bp deletion or insertion distribution is calculated as the percentage of the number of deletions or insertions at each site out of the total number of sequences obtained from sequencing. Deletions or insertions at sites with a SD greater than the mean are considered to be caused by human factors such as PCR and are therefore removed. This invention utilizes a mouse model (IgH) containing the same knock-in sequence in two antibody alleles: a nonfunctional allele and a functional allele. H / 12This study investigated whether the targeting effects of AID on the non-functional and functional alleles of antibodies were consistent. These two antibody heavy chain alleles differ at a 12bp sequence position, which is deleted in the exon of the non-functional allele, thus allowing for differentiation via allele-specific PCR. This invention performed deep sequencing on amplicones of the non-functional and functional alleles of antibodies from GC-B cell DNA and analyzed the effects of AID on these two alleles. The non-functional and functional alleles have similar mutation frequencies, indicating that the targeting effects of AID on the two antibody alleles are identical. Figure 2 B). Next, this invention examined the deletion and insertion frequencies of two antibody alleles (Pas-Ig and Pas-Ig), finding that the deletion frequency of the non-functional antibody allele (Pas-Ig) was higher than that of the functional allele (Pas-Ig). Figure 2 C). This is because the antibody non-functional allele is able to capture more frameshift deletion events. Notably, using an optimized protocol, the antibody non-functional allele also showed a higher insertion frequency than the functional allele, although it was an order of magnitude lower than the deletion frequency. Figure 2 (D) Similar to deletions, the non-functional antibody allele captured more frameshift insertion events than the functional allele. Given the sequence consistency between the non-functional and functional alleles, this invention assumes that human error during the experiment has the same probability of occurring in both alleles. The experimental results of this invention demonstrate that the non-functional antibody allele can capture both deletions and insertions of antibody genes generated during actual SHM processes. In summary, these results indicate that the optimized experimental system can detect both deletions and insertions of antibody genes, particularly insertion events, which occur at extremely low frequencies.

[0054] Example 2.1 bp gene deletions and insertions are the most common gene deletion and insertion events.

[0055] To further investigate the characteristics of antibody gene deletions and insertions, this invention analyzed the frequency of gene deletions and insertions of various lengths. Notably, 1 bp gene deletions and insertions were the most common events, accounting for 45% of all deletions or insertions in non-functional antibody alleles. Figure 2 EF), which leads to a large proportion of frameshift mutations; 80% of gene deletions or insertions result in frameshift mutations. If 1bp gene deletions or insertions are excluded, the proportion of frameshift mutations is consistent with the predicted proportion (assuming that deletions and insertions of different sizes occur at the same frequency, then two-thirds are frameshift mutations). Figure 2In summary, these data indicate that 1bp deletions and insertions are frequent events, leading to a greater predisposition to B-cell-detrimental mutations during SHM. In functional alleles, 1bp deletions and insertions were negatively selected out due to their detrimental effect on BCR. These findings in antibody-nonfunctional alleles suggest that the mechanisms underlying 1bp gene deletions and insertions may differ from those underlying long-fragment deletions and insertions (further details on the mechanisms are provided below).

[0056] This invention further investigates whether the 1bp deletion and insertion events observed in antibody nonfunctional alleles are true results of AID action. First, the frequency of 1bp deletions and insertions in antibody nonfunctional alleles is significantly higher than in functional alleles with the same sequence (average 1bp deletion events per mouse are 87,000 and 13,000 in antibody nonfunctional alleles and 6,000 and 800 in functional alleles, respectively), indicating that under the same PCR and sequencing conditions, antibody nonfunctional alleles can capture true 1bp deletion and insertion events above background levels. Second, the types of deleted or inserted nucleotides differ. Most deleted nucleotides are C:G, while inserted nucleotides are mainly A:T (…). Figure 2 Third, the hotspot locations for 1bp gene deletion and 1bp gene insertion differ in the non-functional alleles of the antibody, while these hotspot locations were not found in the functional alleles. Figure 2 KL). 1bp deletions tend to occur at SHM hotspots and along a series of identical bases, while 1bp insertions occur near deletion hotspots. Finally, and more importantly, no 1bp deletions or insertions were found in the antibody-nonfunctional alleles in AID knockout GC B cells, indicating that 1bp deletions and insertions occur during the AID-initiated SHM process. Figure 2 Therefore, 1bp deletions and insertions generated by AID are common real-world events and products of the SHM process. The different distribution characteristics suggest that the generation mechanisms of 1bp deletions and 1bp insertions may be different (see below for details).

[0057] Next, this invention examines whether the frequency of long gene deletions and insertions in antibody non-functional and functional alleles matches theoretical predictions. Compared to antibody functional alleles, non-functional alleles captured approximately twice as many gene deletions and insertions between 2 bp and 20 bp in size, which is associated with the functional alleles lacking two-thirds of frameshift deletions and insertions. For deletions greater than 20 bp, the increase in non-functional alleles compared to functional alleles is more than two-fold, indicating that deletions greater than 20 bp are detrimental to the production of functional antibodies and are therefore negatively selected and eliminated in functional alleles. Notably, in functional alleles, short deletions (2–20 bp) rather than long deletions (greater than 20 bp) typically occur in CDR regions rather than frame regions. This result suggests that although gene deletions occur frequently, deletions in frame regions and deletions longer than 20 bp in CDR regions are negatively selected and eliminated. On the other hand, for gene insertions longer than 20 bp, both non-functional and functional allele-captured gene insertion events are rare, indicating that insertions longer than 20 bp are inherently infrequent. Therefore, insertions longer than 20 bp have a very low probability of occurring during the SHM process.

[0058] Next, this invention investigated the proportion of deletion and insertion events captured on non-functional antibody alleles. Based on the above analysis, these deletion and insertion events may contribute to increasing antibody diversity. Although gene deletions accounted for 94% of all deletion and insertion events, only 8% of deletions are likely to produce functional antibodies if we consider that deletions cannot alter the reading frame and cannot exceed 20 bp. Figure 2 (O). The remaining 6% were insertion events, of which only 1% of insertions did not alter the reading frame and could potentially generate functional antibodies. Therefore, the total number of deletions and insertions that could potentially generate functional antibodies was less than 9%, a proportion far lower than the expected 33% (assuming that deletions and insertions of all lengths occurred at the same frequency). This result is consistent with the scarcity of deletions and insertions, especially insertions, that could generate functional antibodies in the antibody library. In summary, by analyzing deletions and insertions of non-functional alleles of antibodies and comparing them with functional alleles, this invention found that 1 bp gene deletions and insertions are the most common deletions and insertions, while long-fragment deletions and insertions that do not alter the reading frame and contribute to the generation of functional antibodies are very rare.

[0059] Example 3. The insertion of adjacent repeat sequences generated during the SHM process can produce long CDR3.

[0060] To further study the characteristics of rare gene insertions, this invention develops an algorithm to calculate the similarity score between the inserted sequence and its neighboring sequences. This involves comparing the inserted sequence with its left and right neighboring sequences, counting the number of identical bases at the same position, dividing this number by the total number of bases in the inserted sequence, and taking the maximum percentage as the similarity score. Figure 3 A). This invention has found that in both non-functional and functional antibody alleles, approximately 90% of long fragment insertions show a similarity of over 60%, with the majority of these similarities ranging from 90% to 100%. Figure 3 (B) These results indicate that most insertions are repetitions of adjacent sequences. This invention analyzed the similarity of gene insertions in broadly neutralizing anti-HIV antibodies and found that most insertions had a similarity between 60-80%, accompanied by a large number of point mutations. These results suggest that insertions in broadly neutralizing anti-HIV antibodies can be generated through the insertion of adjacent repetitive sequences produced during the SHM process.

[0061] This invention further analyzes the insertion of these repetitive sequences in detail. Over 70% of the repetitive sequence insertions have micro-homologous sequences between the copied sequence and adjacent sequences. Figure 3 The CD (Continuous Duplicate) sequence indicates that the insertion of repetitive sequences may be generated through a micro-homologous sequence-mediated end-joining mechanism. Furthermore, this invention analyzes the location of repetitive sequence insertions, finding that they mainly occur in the CDR region overlapping with SHM hotspots, which differs from the location of 1 bp insertions, suggesting that the mechanisms underlying long-fragment insertions and 1 bp insertions may differ. Figure 3 E)(For a detailed explanation of the mechanism, please see below).

[0062] This invention investigated whether the insertion of repetitive sequences in a mouse model contributes to the generation of long CDR3. The length of the antibody gene CDR3 in knock-in mice is fixed, consisting of 11 amino acids, with the sequence YDYYGSSYFDY (as shown in SEQ ID NO. 20). This invention found that the insertion of repetitive sequences generated during SHM can lengthen CDR3 and increase CDR3 diversity. Figure 3 F). First, the diversity of insertion length determines the diversity of CDR3 length. For example, the insertion of a 21bp repeat sequence can extend CDR3 from 11 amino acids to 18 amino acids. Figure 3 F, Examples 1-5). Secondly, point mutations occurring in the inserted sequence increase the diversity of CDR3 (F, Examples 1-5). Figure 3 F, Examples 2, 4, 5). Third, when a repeating sequence is inserted that contains a sequence outside the CDR3 region, the diversity of CDR3 also increases. Figure 3F, Example 3). Fourth, repetitive sequence insertions occurring in reading frames 2 or 3 may introduce mutated amino acids at the insertion junction, thereby further increasing the diversity of CDR3 (F, Example 3). Figure 3 F, Examples 4-5). In summary, the experimental results of this invention demonstrate that long CDR3 can be generated during the SHM process. Based on the results obtained from mouse models according to this invention, long CDR3 in broadly neutralizing antibodies may be generated through the insertion of repetitive sequences during the SHM process. Indeed, some broadly neutralizing antibodies with long CDR3 have potential repetitive sequence insertions in their CDR3 that are generated during the SHM process, such as VRC08. VRC08 is a potent broadly neutralizing antibody against HIV, and its unmutated original sequence (UCA) has recently been deduced ( Figure 3 G). In summary, the data from this invention demonstrate that the insertion of repetitive sequences can increase the length of CDRs (including CDR3) during antibody diversification in response to viral infection.

[0063] Besides broad-spectrum neutralizing antibodies, antibodies with long CDR3s are also present in B-cell repertoires of autoimmune diseases, such as systemic lupus erythematosus (SLE). To determine whether the long CDR3s in the SLE antibody repertoire were generated during SHM (Self-Solving Method), this invention analyzed antibody repertoires from eight SLE patients. After classifying antibody sequences according to JH type (including the shortest JH4), this invention found that the average CDR3 length of AID-treated B cells (unmutated V region gene, IgG+ or IgA+) was significantly increased compared to resting B cells (unmutated V region gene, IgM+). Therefore, CDR3s in the SLE antibody repertoire increase during SHM. Further studies revealed the presence of a 42bp continuous repeat sequence in this antibody repertoire. In summary, the analysis of mouse and human data in this invention demonstrates that the insertion of repeat sequences generated during SHM can lengthen the CDR region sequence and contribute to the generation of long CDR3s specific to broad-spectrum antiviral neutralizing antibodies and autoantibodies.

[0064] Example 4. Antibody diversification analysis in a mouse model of DNA damage repair factor knockout antibody nonfunctional alleles.

[0065] To further elucidate the mechanisms of antibody gene deletion and insertion, this invention utilized CRISPR-Cas9 gene editing technology to construct a series of DNA damage repair factor knockout mice. These knockout mice were then mated with antibody non-functional allele mice to obtain DNA damage repair factor knockout mouse models (IgH) carrying antibody non-functional alleles. WT / 12 X - / -This invention analyzed 12 DNA damage repair factor knockout mouse models carrying antibody-free nonfunctional alleles and categorized them according to repair mechanisms: the Ung-initiated BER pathway, containing Ung and Fen1 proteins; the Msh2-initiated MMR pathway, containing Msh2, Pms2, and Mlh1 proteins; the c-NHEJ pathway's core protein XLF; DSB damage stress proteins, including ATM and 53bp1; the DNA transdamage replicase polη; and the DNA exonuclease-active proteins APE2 (located on the sex chromosome and encoded by Apex2) and Exo1. To further confirm the correctness of the gene knockout mice constructed in this invention, the relevant phenotypes of these knockout mice were analyzed, including B cell development, the number and proportion of GC B cells, CSR levels, and SHM patterns.

[0066] Consistent with previous reports, the number of B cells was reduced to varying degrees in some knockout mice, primarily Apex2 knockout mice. - / - 53bp1 - / - Xlf - / - , and Atm - / - Mice. Normal wild-type mice, through the above immunization strategy, can obtain 10 6 Orders of magnitude greater than GC B cells, while at Atm - / - In mice, only less than 4x10 can be obtained. 5 GC B cells ( Figure 4 A). Clearly, the absence of ATM affects the germinal center response and reduces the number of GC B cells, which is consistent with previous reports, due to the 53bp1... - / - Xlf - / - Apex2 - / - In mice, B cell development and survival were impaired, resulting in varying degrees of suppression of the germinal center response and a decrease in the number of GC B cells in these gene knockout mice. Regarding the Fen1 gene, this invention did not obtain homozygous Fen1. - / - In mice, consistent with previous reports, Fen1 knockout mice exhibited an embryonic lethal phenotype. Other genotype knockout mice did not show defects in B cell development, nor were germinal center responses or GC B cell numbers affected.

[0067] Next, this invention analyzed the SHM profiles of these gene knockout mice, including mutation frequency, mutation sites, and mutated base types. Apex2 - / Y Apex2 - / - Atm - / - The overall mutation frequency in mice was significantly reduced. Figure 4 (BD), which is consistent with previously reported results. And in Mlh1 - / -Pms2 - / - Exo1 - / - In mice, the overall decrease in mutation frequency was due to changes in the mutation profile. Mlh1, Pms2, and Exo1 are important molecules in the MMR pathway, and their deletion reduces the mutation frequency at A:T base sites and the transversion mutation frequency at C:G base sites. Figure 4 BD). Msh2 - / - and Pohl - / - The mutation frequency at the A:T site was significantly reduced in mice. Figure 4 BD). Ung - / - and Ung - / - Msh2 - / - Transition mutations decreased in mice, but C:G site transition mutations increased, which is consistent with previous reports. Figure 4 BD). In addition, Ung - / - Msh2 - / - Ung - / - and 53bp1 - / - CSR levels in mice were significantly reduced, Atm - / - Xlf - / - Msh2 - / - Mlh1 - / - and Exo1 - / - The CSR level in mice was also slightly reduced. Figure 4 E). In summary, the SHM and CSR phenotypes of the DNA damage repair factor knockout mice carrying antibody nonfunctional alleles constructed in this invention are consistent with those of the previously reported DNA damage repair factor knockout mice without antibody nonfunctional alleles.

[0068] Example 5. Different mechanisms mediate the generation of 1bp and long fragment gene deletions and insertions.

[0069] After confirming that the mutation patterns in the gene knockout mouse model of this invention were consistent with expectations, this invention further analyzed the deletion and insertion events of non-functional alleles of antibodies in these mice. - / - Msh2 - / - The deletion and insertion of antibody genes were significantly reduced in mice, indicating that the generation of deletions and insertions depends on the BER and MMR pathways. - / - In mice, both 1bp and long fragment deletion events were significantly reduced, while 53bp deletion events were significantly reduced. - / - The frequency of long fragment deletions was significantly reduced in mice, with little effect on the frequency of 1bp deletions. Figure 5 A). These results indicate that the generation of long fragment deletions requires additional regulatory factors, such as 53bp1, which may be related to the function of 53bp1 in inhibiting DNA end splicing. Exo1 - / -and Apex2 - / - The frequency of 1bp deletion was reduced in mice, indicating that the nuclease activities of Exol and Ape2 can promote the generation of 1bp deletion. Figure 5 A). On the other hand, the 1bp insertion frequency is only in Msh2. - / - Slightly decreased in mice, in Ung - / - No significant changes were observed in mice, indicating that a 1bp insertion can be generated through both BER and MMR pathways. Figure 5 B). The generation of a 1bp insertion requires Exo1 and Polη, a result consistent with the fact that the hotspot location of the 1bp insertion is not at the AID lesion, but rather near the lesion. Figure 2 L). Exo1 is typically recruited upstream or downstream of DNA mismatches, while Po1η promotes A:T base insertion. Conversely, long fragment insertions depend on 53bp1, Exo1, and X1f, all of which are downstream molecules of Ung in the BER pathway. Figure 5 B). Since Polη does not affect the generation of long fragment insertions, then another DNA polymerase must be involved in the generation of long fragment insertions.

[0070] To identify DNA polymerases mediating long fragment insertions, this invention constructed six knockout CH12 B cell lines containing DNA polymerases: Polβ (encoded by the gene Polb), Polλ (encoded by the gene Poll), Polμ (encoded by the gene Polm), Polι (encoded by the gene Poli), Polk (encoded by the gene Polk), and Polθ (encoded by the gene Polq). This invention overexpressed AID in these mutant cell lines and wild-type cell lines, comparing mutations, deletions, and insertions in their antibody heavy chain gene V(D)J. Using deep sequencing and established analytical methods for nonfunctional allele mutations, deletions, and insertions in mouse antibodies, this invention performed a detailed analysis of mutations, deletions, and insertions in the V(D)J region of the B cell lines. - / - In these cells, the insertion frequency of long fragments increased significantly, while the mutation and deletion frequencies did not change significantly. Figure 5 Further analysis revealed that these gene insertions were primarily insertions into adjacent repetitive sequences, consistent with the insertion types observed in GC B cells. In Poll... - / - In these cells, the insertion frequency of long fragments was significantly reduced, while the mutation and deletion frequencies did not change significantly, which is consistent with that in Polb. - / - The phenomenon observed in the cells is exactly the opposite ( Figure 5In cells knocked out Polμ, Polι, Polκ, and Polκ, the mutation, deletion, and insertion frequencies of V(D)J did not change significantly. In summary, this invention reveals that Polλ is a DNA polymerase that mediates AID-dependent long fragment insertions, while Polβ can inhibit the generation of these long fragment insertions.

[0071] Next, this invention further analyzed the frequency of deletions and insertions of various fragment sizes in different gene knockout mice. This invention found that knockout of Msh2, Mlh1, and Pms2 in the MMR pathway resulted in an increase in the frequency of 4bp deletions and 3bp insertions. Figure 5 These deletion and insertion events occur at locations in the CDR3 region containing a 3-base tandem repeat (GTAGTA). Figure 5 These results indicate that during SHM, the MMR pathway factors Msh2, Mlh1, and Pms2 inhibit the generation of deletions and insertions at antibody gene tandem repeat sequences. This is likely because, in MMR pathway defects, slip mismatches of DNA polymerase at AID lesions lead to the introduction of gene deletions and insertions at these short tandem repeat sequences. This phenomenon has been observed in microsatellite regions with longer tandem repeat sequences (5-50 bp tandem repeats). This invention reveals a sequence-dependent role of the MMR pathway in promoting gene deletions and insertions during SHM. This invention also suggests that at the genome-wide level, MMR defects can promote the introduction of gene deletions and insertions in regions with only one tandem repeat sequence.

[0072] Example 6. Construction of a mouse model with increased antibody gene deletion or insertion frequency

[0073] In these genetically modified mouse models, this invention seeks to identify models that can increase gene deletions and insertions without altering reading frames, which could be used for vaccine research and broad-spectrum neutralizing antibody screening. This invention analyzes the frequency of gene deletions and insertions at 3-fold increases in antibody non-functional alleles in mice with different gene knockouts. This invention found that only Msh2... - / - Mice can increase the frequency of potentially functionally lost segments. Figure 5 K), however Msh2 - / - The significant reduction in A:T mutations in mice may limit the diversity of the antibody repertoire. Furthermore, the frequency of potentially functional insertions was not significantly increased in these gene-deleted mice. Figure 5 L).

[0074] Next, this invention constructed a mouse model capable of increasing the frequency of antibody gene deletions and insertions without altering the mutation profile of the antibody library. Trex2 possesses exonuclease activity in the 3' to 5' direction and can participate in the processing and repair of DNA end damage. This invention constructed a mouse model of antibody-nonfunctional alleles that specifically overexpress Trex2 in mature B cells and tested the experimental mice (Rosa26). + / Trex2 Cd21 Cre IgH WT / 12 ) and control mice (Rosa26) + / + Cd21 Cre IgH WT / 12 The frequency of mutations, deletions, and insertions in the nonfunctional alleles of the antibody gene was determined. This invention found that a mouse model overexpressing Trex2 significantly increased the deletion frequency of the antibody gene, while the mutation frequency, mutation profile, insertion frequency, and CSR showed no significant changes compared to the control group. Figure 6 Further analysis revealed that the Trex2 overexpression mouse model primarily increased the frequency of long-fragment deletions, with no effect on the frequency of 1bp deletions. Figure 6 (B) Furthermore, in the Trex2 overexpression mouse model, the frequency of potentially functional gene deletions, i.e., in-frame deletions of 3-18 bp, was doubled compared to the control group. In summary, this invention provides a mouse model that can increase the frequency of antibody gene deletions.

[0075] On the other hand, in in vitro B cell line experiments, this invention has found that the insertion frequency of long fragments is significantly increased in Polb- / - cells. Based on this experimental result, this invention constructed a mouse model of specifically knocking out Polβ in GCB cells. This invention found similar results to those in Polb- / - cells, in Po1β-knockout GCB cells, the insertion frequency of long fragments greater than 8 bp was significantly increased (…). Figure 6 (DF). Therefore, this invention provides a mouse model that can increase the frequency of antibody gene insertion. These two mouse models, which can increase the deletion or insertion of antibody genes, can be used in conjunction with humanized mouse models to increase the level of deletion or insertion of antibody genes in the initially activated B cell library, or to increase the level of long CDR3, thereby improving the screening efficiency of rare antibodies.

[0076] Gene deletions and insertions are byproducts of antibody diversification, and their widespread attention was only recently drawn to their existence and important role in rare broad-spectrum antiviral neutralizing antibodies. This invention analyzed the characteristics of gene deletion and insertion events generated during the antibody-derived non-functional allele mouse process (SHM). The invention found that deletion and insertion events that produce functional antibodies are rare, with harmful 1bp deletions and insertions dominating all deletion and insertion events. The invention then systematically dissected the different mechanisms generating 1bp and longer fragment deletion and insertion events. Finally, the invention constructed two potential mouse models for screening broad-spectrum neutralizing antibodies with gene deletions or insertions through genetic modification.

[0077] Many reported gene knockout models have helped to understand downstream DNA repair pathways of AID, revealing the mechanisms underlying different mutational profiles and antibody type switching during SHM. However, the mechanisms underlying gene deletions and insertions remain unclear. This invention overcomes the difficulties in detecting deletions and insertions by utilizing an antibody nonfunctional allele system, and further constructs different gene knockout mice within this model to elucidate the mechanisms of antibody gene deletions and insertions during SHM. As summarized in the radar chart, this invention reveals the outcomes of different repair pathways following AID action, including previously undetectable and overlooked deletion and insertion events. Figure 6 G). These data enabled the present invention to successfully analyze how different DNA repair pathways generate 1 bp and long fragment deletion and insertion events, and to explain why certain deletion and insertion events are more prevalent. Figure 7 AB).

[0078] The BER and MMR pathways are two major DNA repair pathways that translate AID damage into different mutational outcomes. BER is responsible for gene deletions, while gene insertions can occur in both the BER and MMR pathways. Ung and Msh2 are two key factors in the BER and MMR pathways, respectively, and their effects on gene deletions are diametrically opposed. Deletion events are significantly reduced in Ung- / - mice, while they are significantly increased in Msh2- / - mice. This is consistent with the concept that Ung and Msh2 work together to maintain a balance between the downstream BER and MMR pathways in AID damage. Therefore, impairment of either pathway will cause AID damage repair to shift to the other.

[0079] Downstream of Ung, the repair mechanism for AID damage differs. 53bp1 - / -In mice, the mutation profile and 1bp gene deletions and insertions were not significantly affected, but antibody type switching and long fragment deletions and insertions were significantly affected, indicating that antibody type switching and long fragment deletions and insertions may occur in a common pathway involving 53bp. Long fragment insertions are more difficult to generate than long fragment deletions because this process requires the participation of certain DNA polymerases. This invention found that the DNA polymerase Polλ is involved in the generation of long fragment insertions.

[0080] This invention is significant for generating animal models for testing HIV vaccines and inducing broadly neutralizing antibodies. In recent years, broadly neutralizing antibodies isolated from HIV-infected patients have rekindled hopes for HIV vaccine development. These anti-HIV broadly neutralizing antibodies possess unique properties, such as the use of specific germline V genes, high levels of point mutations, frequent deletions and insertions, long CDR3, and self / multireactivity. Based on the unique properties of anti-HIV broadly neutralizing antibodies, many artificially modified mouse models have been applied to vaccine development. Previous studies have shown that animals carrying the human germline V gene... H The 1-2 mouse model can be randomly linked with mouse Ds and Js to form different CDR H3s, generating antibodies that can neutralize multiple strains. The generation of broad-spectrum neutralizing antibodies depends on two steps: activation of resting B cell precursors and antibody affinity maturation. To screen for BCRs with gene deletions and insertions, the original BCR library after AID activation needs to contain gene deletions and insertions. Therefore, increasing the frequency of gene deletions and insertions in the original BCR library helps improve the likelihood of screening for antibodies with gene deletions and insertions. The Trex2 overexpression mouse model in this invention can promote the generation of gene deletions, and the Polβ conditional knockout mouse model can promote the generation of gene insertions. These two mouse models can be used in conjunction with the aforementioned humanized antibody mouse model for vaccine development and the production of broad-spectrum neutralizing antibodies.

[0081] Furthermore, data from this invention show that the insertion of adjacent repetitive sequences during SHM can generate long CDR3, an antibody characteristic previously thought to be primarily generated during V(D)J rearrangements. The mouse model of this invention overcomes issues concerning CDR3 diversity and the rarity of gene insertion, revealing that AID-mediated repetitive sequence insertion contributes to the generation of antibodies carrying long CDR3. B cells with long CDR3 in the BCR are likely to be autoreactive / multireactive and are typically eliminated by negative selection. Therefore, a pool of immune-tolerant activated B cells may be a potential source of broad-spectrum neutralizing antibody precursors carrying long CDR3. On the other hand, considering that antibodies obtaining long CDR3 through repetitive sequence insertion during SHM may be autoreactive / multireactive, the results of this invention suggest that SLE may be caused by the SHM process.

[0082] Finally, while this invention focuses primarily on the mechanisms of deletions and insertions during AID damage repair, these mechanisms may also apply to other types of DNA damage repair, including damage resulting from CRISPR base editing. The downstream repair pathways for these DNA damages are generally the same. Gene deletions and insertions frequently occur in cancer genomes, and deficiencies in many DNA damage repair factors are associated with increased deletions and insertions in cancer cells. The underlying mechanisms of different classes of mutations and variations in the human genome remain an important question in cancer genetics and human disease research. This invention addresses the impact of loss of function of many cancer-related DNA repair factors on rare mutational events, and these results may also explain the mechanisms of oncogene mutations. The results show that deletions and insertions of 1 bp genes are prevalent, indicating that deletions and insertions in the genome are more destructive than previously thought. In AID-initiated cancers (such as diffuse large B-cell lymphoma), mutations in MMR genes can increase the likelihood of harmful gene deletions. Furthermore, the results of the MMR pathway-related gene knockout mouse model of this invention suggest that tandem repeat sequences as short as two copies may be sites prone to deletions and insertions. Such events can occur at the genome-wide level in MMR-deficient cells and may trigger cancer.

[0083] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for detecting extremely low-frequency insertion events, characterized in that, The method includes the following steps: Immunizing a mouse model with non-functional alleles of the antibody using sheep blood and adjuvant 2'3'-c-diAM(PS)2(Rp,Rp) on days 0 and 6, respectively; harvesting the spleens of mice on day 9 post-immunization, purifying germinal center B cells using a B cell purification kit combined with magnetic bead sorting, adding biotin-labeled antibodies CD43, CD11c, and IgD, and removing non-germinal center B cells using magnetic bead sorting to obtain 1 million cells; extracting genomic DNA from GC B cells, performing PCR amplification using primers specific to non-functional alleles, constructing a sequencing library, and performing high-throughput sequencing to ensure that each PCR amplicon reaches a sequencing depth of 5 million sequences; this method can detect and obtain gene insertion events with a frequency as low as one in a thousand. In the antibody non-functional allele mouse model, the antibody heavy chain allele includes one functional allele and one non-functional allele. The functional allele successfully expresses the B cell receptor, maintaining B cell survival. The non-functional allele is a gene sequence VHB1-8 inserted into the antibody heavy chain region, with a 2bp, 12bp, 14bp, or 47bp deletion at the 3' end, thus not encoding a protein and not subject to selection pressure. Both the functional and non-functional alleles are transcribed through the same promoter. The gene sequence of the nonfunctional allele of the antibody is shown in SEQ ID NO.1-4.

2. A bioinformatics analysis method for antibody gene deletion and insertion, characterized in that, The method includes the following steps: performing high-throughput sequencing according to the method of claim 1, and aligning the sequencing results with the Bowtie2 and B1-8 reference sequences; if the sequencing results have a redundant sequence compared with the reference sequence, and the Illumina sequencing quality score of no two consecutive nucleotides in the sequencing results is less than 20, then the redundant sequence is considered a gene insertion event; if the sequencing results have a gap compared with the reference sequence, and the Illumina sequencing quality score of the four consecutive nucleotides on both sides of the gap is greater than 20, then the gap is considered a gene deletion event. For gene deletion and insertion events, a comprehensive and rapid analysis is conducted in terms of frequency, fragment size, and distribution characteristics. Specifically, this includes: analysis of total deletion or insertion frequency, analysis of deletion or insertion distribution characteristics, analysis of deletion or insertion frequency of different fragment sizes, and analysis of 1bp deletion or insertion distribution characteristics. And / or, the similarity between the inserted base fragment and the bases of the neighboring sequence is evaluated, and the source characteristics of the inserted fragment are defined by calculating the similarity score. The method is to compare the inserted sequence with the neighboring sequences on the left and right sides, count the number of bases that are consistent at the same position, divide the number by the total number of bases in the inserted sequence, and take the maximum percentage, which is the similarity score.

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