CN112877314A

CN112877314A - Inducible base editing system and application thereof

Info

Publication number: CN112877314A
Application number: CN202110249394.4A
Authority: CN
Inventors: 姚少华; 龙洁
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-03-08
Filing date: 2021-03-08
Publication date: 2021-06-01
Anticipated expiration: 2041-03-08
Also published as: CN112877314B

Abstract

The invention discloses an inducible base editing system and application thereof. The base editing system comprises inducible human-derived cytosine deaminase (A3A) and single-cut active SpCas 9; deaminase can be resolved based on potential amino acid sites; rapamycin reassembles the cleaved deaminase by interacting with the N-and C-termini of the amino acid cleavage site via FRB/FKBP and induces complete base editing. The base editing activity of the gene is controlled by constructing an inducible base editing system, so that off-target editing is reduced; at the same time, the split design in this application does not reduce targeted editing and therefore can be used as a compensation strategy for protein mutations and can be used in conjunction with these mutated deaminases to further reduce off-target editing.

Description

Inducible base editing system and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an inducible base editing system and application thereof.

Background

Base editing is a genome editing strategy that can directly convert specific single nucleotides in genomic DNA (Rees and Liu 2018). Currently, there are two types of base editors, Cytosine Base Editor (CBE) and Adenine Base Editor (ABE), both of which consist of deaminase and a catalytic deficient Cas9 protein, which can catalyze deamination of cytosine or adenine. Under the direction of the sgRNA, the base editor can convert C-G base pairs to T-a base pairs, or a-T base pairs to G-C base pairs, within a specific window of base pairs within the target. Since it is highly efficient and accurate and does not generate by-products such as insertions or deletions introduced by DNA Double Strand Breaks (DSBs), the technique is rapidly being widely used in various model organisms such as mouse, zebrafish, Arabidopsis thaliana and Saccharomyces cerevisiae.

Although base editing techniques have achieved many exciting results in extensive basic research, there is still a major impediment to base editing in clinical applications, namely off-target effects at the DNA or RNA level. Current CBE and ABE often cause off-target in RNA in the whole transcriptome range, raising concerns about safety. More seriously, since CBE also causes off-target at the DNA level, it may further lead to pathological conditions including cancer. Indeed, as early as thirty years ago, researchers found that overexpression of APOBEC1 (one of the most prevalent cytosine deaminase used in CBE) could lead to liver cancer in transgenic mice. Subsequently, AID was also found to induce tumorigenesis when overexpressed in mice. Based on these findings, an increasing number of studies have demonstrated the association of cytosine deaminase with tumorigenesis in a variety of tissues. Members of the cytosine deaminases found, such as APOBEC3B, are in a variety of solid tumors.

It has been found that overexpression of cytosine deaminase can induce base substitutions in genomic DNA, thereby compromising genomic stability. More importantly, many tumor cells have been found to carry SNVs characterized by cytosine markers. Therefore, uncontrolled base editors (CBEs in particular) may give rise to tumors. Given that AAV is one of the most effective in vivo delivery vectors currently available, and that long term expression of transgenes in vivo following AAV injection, the use of such vectors to deliver base editors would clearly amplify off-target consequences. Therefore, there is an urgent need to explore novel base editing tools with controllable activities.

Disclosure of Invention

In view of the above-mentioned disadvantages in the prior art, the present invention provides an inducible base editing system and its application, by which editing time can be expected to be shortened by constructing a split base editing system and controlling its base editing activity by induction of rapamycin. At the same time, the split design in this application does not reduce targeted editing and therefore can be used as a compensation strategy for protein mutations and can be used in conjunction with these mutated deaminases to further reduce off-target editing.

In recent years, many studies have shown the off-target effects of DNA and RNA present in base editors. The invention controls the editing activity of the base editor by controlling the activity of the deaminase, thereby reducing the off-target effect. The base editing system of the invention comprises inducible human cytosine deaminase (A3A) and single-nicked active SpCas 9. The specific scheme is as follows: after A3A is cleaved into inactive N-and C-termini, which are fused to FRB and FKBP proteins, respectively, FRB and FKBP form stable ternary complexes under rapamycin induction, allowing the deaminase to reassemble into functional A3A. Since deaminase activity is regulated by an inducer, the activity of the base editor is thus controllable.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

an inducible base editing system comprising a base editor, and rapamycin associated therewith; the base editor comprises a deaminase;

deaminase can be split based on any amino acid site; rapamycin is combined with deaminase through the split amino acid site and induced to complete base editing.

Further, the base editor is a base editor consisting of a deaminase and BE3/SpCas 9.

Further, FRB and FKBP of rapamycin are coupled to the cleavage of the nonstructural loop of the deaminase amino acid cleavage site, respectively.

Further, FRB and FKBP are fused with the N-terminal and C-terminal of the deaminase amino acid splitting site, respectively, and dimerize to form heterodimers, completing the assembly of the base editor.

Further, the resolution site of deaminase is amino acid 44, 85, 118 or 147.

Further, the resolution site of deaminase is amino acid 85.

Further, the deaminase is a cytosine deaminase.

Further, the cytosine deaminase is APOBEC3A cytosine deaminase or APOBEC1 cytosine deaminase.

Further, the concentration of rapamycin is 0.01-200 nM.

Further, the concentration of rapamycin was 200 nM.

The use of the above-described inducible base editing system for gene editing.

A kit for gene editing comprising the inducible base editing system.

The base editing system constructed in the application can keep Cas9 intact, so that the system can BE compatible with other non-traditional base editors, such as CP-Cas9 derived base editors (Huang et al, 2019), internally-mosaiced BE-PIGS base editing (Wang et al, 2019) and sgRNA framework modified sgBEs (Wang et al, 2020).

The invention has the beneficial effects that:

according to the scheme, a split base editing system is constructed, the base editing activity of rapamycin can be controlled through induction, and the editing time can be expected to be shortened. At the same time, the split design in this application does not reduce targeted editing and therefore can be used as a compensation strategy for protein mutations and can be used in conjunction with these mutated deaminases to further reduce off-target editing.

Drawings

FIG. 1 is a scheme showing the design of the inducible split-A3A-BE 3;

FIG. 2 shows the results of the detection of inducible editing activity of 4 split-A3A-BE 3;

FIG. 3 is a graph showing the results of examining the influence of the action concentration and duration of rapamycin on the base editing of sA3A-BE 3-85;

FIG. 4 is the feature detection of the editing mode of sA3A-BE 3-85;

FIG. 5 shows the result of DNA off-target editing detection of sA3A-BE 3-85;

FIG. 6 shows the compiled results of background editing for subcellular localization of sA3A-BE3-85 controlled by ERT2 system to reduce background;

FIG. 7 shows the construction process and characteristic detection of split-APOBEC1-BE 3.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

EXAMPLE 1 construction of split-A3A plasmid

A split-A3A plasmid was constructed on the basis of A3A-BE3 to form a series of N-terminal split-A3A and its corresponding C-terminal split-BE3-A3As (FIG. 1 b). As shown in FIG. 1c, 4 groups of split-A3A-BE3(sA3A-BE3) were constructed and named by their amino acid splitting sites, namely, sA3A-BE3-44, sA3A-BE3-85, sA3A-BE3-118 and sA3A-BE3 are-147, respectively.

FIG. 1 is a design of inducible split-A3A-BE 3; wherein, the figure a shows a schematic diagram of inducible split-A3A-BE3 recombination, and rapamycin induces dimerization of FRB and FKBP fused to the N-terminal and C-terminal of A3A respectively to form heterodimer, thereby completing the assembly of a functional A3A-BE3 base editor. FIG. b is a schematic representation of the structure of A3A (PDB: 5 keg). The four nonstructural loops opposite the ssDNA-A3A binding interface are candidate cleavage sites, represented as spheres in the figure. FIG. c is a schematic diagram of the construction of split-A3A-BE3 cytosine base editor.

Example 2 Split-A3A base editing

1. sA3A-BE3 inducible base editing activity assay

In 3 endogenous targets, each pair of split-A3A-BE3 editors was co-transfected with sgRNA into HEK293T cells, respectively. As shown in FIG. 2a, all sA3A-BE3 showed inducible editing activity, although with different potential. Among these sA3A-BE3, sA3A-BE3-44 was the most efficient at editing at all 3 target sites when induced with 200nM rapamycin, a concentration that is frequently used in the split system of FRB/FRBP. However, its activity remained essentially unchanged under non-induced conditions. On average, uninduced sA3A-BE3-44 showed comparable activity to full-length A3A-BE3 (FIG. 2 b). Transfection of the C-terminal part of sA3A-BE3-44 did not result in any detectable editing, indicating that the background editing activity of sA3A-BE3-44 was not due to residual deamination activity of C-terminal A3A, possibly due to self-assembly of deaminase. In contrast, sA3A-BE3-147 exhibited minimal editing activity under both uninduced and induced conditions, approximately 7% to 19% of the activity of full-length A3A-BE3, respectively (FIG. 2 b). sA3A-BE3-85 and sA3A-BE3-118 are induced more efficiently than the two. On average, sA3A-BE3-85 was similar to full-length A3A-BE3 in activity under induction conditions, while sA3A-BE3-118 was about 1.7-fold less active than full-length. Of the four split-A3A-BE3 base editors tested, sA3A-BE3-85 showed the best response effect to rapamycin (FIG. 2 b).

FIG. 2 is the inducible editing activity of 4 split-A3A-BE 3; in the figure a, the editing efficiency of the inducible base editor is measured, and in 3 endogenous targets, full-length plasmids of A3A-BE3 or inducible sA3A-BE3s are co-transfected with sgRNA to HEK293T cells respectively. The base editing efficiency was analyzed by Sanger sequencing and EditR (Kluesner et al.2018). Each experiment was repeated at least three times. Data are presented as mean ± SEM. And b is the average editing efficiency of all editors in the graph a at each target point. Normalization was performed with the editing efficiency of full length A3A-BE 3as 1.

2. sA3A-BE3-85 characterization

The effect of induction time of rapamycin on editing efficiency was examined. HEK293T cells transfected with the sA3A-BE3-85 editor described above were treated with 200nM rapamycin for 3, 6, 12, 24 and 48 hours, respectively. As shown in fig. 3a, c, we observe edits that are time dependent. Interestingly, in the three target sites tested, the editing efficiency induced at 24 hours was similar to that induced at 48 hours for both sites, whereas for the remaining one site, the editing efficiency induced at 24 hours was 40.36% lower than that induced at 48 hours, indicating that the effect of the duration of induction was target specific. Next, we tested the effect of rapamycin concentration on the editing efficiency, treating transfected cells with rapamycin at concentrations ranging from 0.01nM to 200nM (FIG. 3 b). As expected, a dose-dependent effect was observed and the dose-effect curve showed that plateau phase began to reach around 50 nM. (FIG. 3 d).

FIG. 3 is a graph showing the effect of concentration and duration of action of rapamycin on base editing of sA3A-BE 3-85; in this figure, a shows the editing efficiency of sA3A-BE3-85 under different induction times of 200nM rapamycin treatment. Transfected HEK293T cells were treated with 200nM rapamycin and the cells harvested at the corresponding time points. FIG. b is a graph showing the editing efficiency of sA3A-BE3-85 after 48 hours of induction with different concentrations of rapamycin. Transfected HEK293T cells were treated with various concentrations of rapamycin for 48 hours before sequence analysis. The effect of duration of induction (panel c) and intensity (panel d) on sA3A-BE3-85 base editing was summarized by analyzing the editing efficiency of all targetable cytosines per site on average.

Example 3 further characterization of split-A3A

It was examined whether the cleavage of A3A at amino acid 85 changed the base editing pattern of A3A-BE3, including editing window, sequence preference and editing purity of C → T.

To determine the editing window, 3 targets with multiple cytosines within the editing window were selected (fig. 4 a). As shown in FIG. 4a, sA3A-BE3-85 does not significantly shift the position of the editing window or affect the width of the editing window compared to full length A3A-BE 3. Then, we tested whether sA3A-BE3-85 has a different sequence preference than full-length A3A-BE3, since previous studies have shown that the unstructured loops between deaminase helices play an important role in substrate recognition (Salter et al.2016). The 9 targets were divided into four groups based on NC motif (GC, CC, AC and TC) and the sequence preferences of sA3A-BE3-85 and full-length A3A-BE3 were compared (FIG. 4 b). On average, the editing efficiency of sA3A-BE3-85 in all four different motifs was similar to that of full-length A3A-BE 3. The editing efficiencies of sA3A-BE3-85 for full-length A3A-BE3 were relative editing efficiencies, 84.5% for GC, 99.7% for CC, 93.5% for TC, and 95.0% for AC, indicating that there was no significantly altered motif preference for sA3A-BE 3-85. To test the editing purity of C to T, we selected 2 previously characterized sites that tended to switch from C to G in addition to C to T (C6 in HEK2 and C6 in RNF 2). We found that sA3A-BE3-85 had 53.05% higher edibility purity than the original A3A-BE3 and 9.18% higher edibility purity at the site of RNF2, indicating that sA3A-BE3-85 slightly improved the purity of the C → T product with higher C → T efficiency (FIG. 4C).

FIG. 4 shows the characteristics of the editing mode of sA3A-BE 3-85; wherein FIG. a is an edit window comparison of sA3A-BE3-85 and full-length A3A-BE 3; FIG. b is a sequence preference comparison of sA3A-BE3-85 and full-length A3A-BE3, calculating the base editing efficiency of each NC motif in each target; FIG. c is a comparison of the editorial purities of full-length A3A-BE3 and sA3A-BE 3-85.

Example 4 DNA off-target editing of split-A3A-BEs

The fact that CBEs induce sequence-dependent and sequence-independent off-target editing greatly limits their applications. To determine the sequence-dependent off-target editing of sA3A-BE3-85, two well-characterized targets (HEK site4 and EXMI) were selected. As shown in FIG. 5a, sA3A-BE3-85 generally showed lower sequence-dependent off-target editing at all three off-target sites. As the induction time was prolonged, the efficiency of sA3A-BE3-85 editing at off-target sites was gradually increased (FIG. 5 a). The ratio of editing efficiency to off-target efficiency was calculated and found to BE higher for sA3A BE3-85 (4: 1) than for A3A BE3 (2.4: 1) (FIG. 5 b).

sA3A-BE3-85 was then tested for sequence-independent off-target editing by orthogonal R-loop assay, with artificial R-loops induced by transfection of the single nickase sacAS9 and sgRNA at specific genomic sites. Co-transfection of the base editor with the R-loop construct showed that sA3A-BE3-85 showed a significant reduction in off-target editing compared to full-length A3A-BE3 (FIG. 5 c). The above results indicate that sA3A-BE3-85 shows a lower tendency in both sequence-dependent and sequence-independent DNA off-target editing than full-length A3A-BE 3.

FIG. 5 is DNA off-target editing of sA3A-BE3-85, where panel a is the sequence-dependent off-target editing efficiency of sA3A-BE3-85, two gRNAs targeting HEK4 and EMX1 sites, respectively, were used to analyze sequence-dependent off-target editing, and the off-target sites were predicted by sequence similarity and have been described in (Komor et al.2016). Analyzing the average editing efficiency of all targetable cytosines at each site; FIG. b is a graph of relative edit efficiency calculated for target edit versus off-target edit ratio; FIG. c shows the principle of detection by orthogonal R loops and sequence-independent off-target editing detection of sA3A-BE 3-85. Two artificial R loops were generated by transfection of nsaCas9 and the corresponding sgRNA, and their target editing efficiency was determined simultaneously (EXM1)

Example 5 reduction of background editing of split-A3A by controlling subcellular localization

Since split-A3A-85 showed background editing when not induced by rapamycin, we attempted to test whether it was possible to further reduce background editing by modulating the subcellular localization of the split-A3A-85 component, thereby spatially separating them when induction was not required.

The nuclear transport system ERT2 was used to control nucleic acid transport at the N-terminal portion of split-A3A-85 so that the N-terminal split-A3A-85 was located in the cytoplasm and was spatially separated from its C-terminus, blocking self-assembly (FIG. 6 a). As shown in FIG. 6b, fusion of the ERT2 domain to the N-terminus, but not the C-terminus, of N-split-A3A-85 achieved inducible editing under induction and significantly reduced background editing in the absence of induction.

FIG. 6 control of the subcellular localization of sA3A-BE3-85 with ERT2 system reduces background editing; wherein panel a shows the mechanism of nuclear transport of sA3A-BE3-85 controlled by ERT2 system, the N-terminal part of sA3A-BE3-85 is fused to ERT2 and transferred into the nucleus induced by 4-OHT, where it is close to the C-terminal part of sA3A-BE3-85 and assembles a functional base editor in the presence of rapamycin; FIG. b is the construction of sA3A-BE3-85 construct induced by the dual control system of 4-OHT and rapamycin; FIG. c is a background editing assay of split-A3A-BE3-85 fused to ERT 2.

Example 6 design and characterization of rat APOBEC 1-derived base editor

After establishing the split-A3A-BE3 system, we next attempted to extend this finding to another widely used cytosine deaminase, rAPOBEC1, which is also often used in base editing. By sequence alignment, rAPOBEC1 has 48.1% similarity to A3A, and the key amino acids responsible for catalytic deamination remain substantially conserved between the two cytosine deaminases. Since the crystal structure of rAPOBEC1 has not been resolved, we predicted its structure from the protein sequence using an online program (MPI biologics Toolkit: https:// Toolkit. tuebingen. mpg. de.). The predicted structure of rAPOBEC1 is similar to A3A, in particular loop 4 and nearby structures in A3A are highly homologous to the corresponding regions in rAPOBEC 1. As shown in FIGS. 7a and b, we mapped the 85 th amino acid of A3A to the 77 th amino acid of rAPOBEC1 by structural comparison and sequence alignment. The resulting split-rAPOBEC 1-BE3 achieved 85.83% of the activity of full-length BE3 under induction conditions, but its activity was significantly reduced without interference, in agreement with the previous results for sA3A-BE3-85 (FIG. 7 c). Thus, the results indicate that our resolution strategy can also be applied to other deaminases.

FIG. 7 shows the design and characteristics of split-APOBEC1-BE 3; wherein, the figure a is the amino acid sequence alignment of APOBEC3A and APOBEC 1; panel b is a structural comparison of A3A and APOBEC1, with the MPI bioinformatics kit predicting the structure of APOBEC1, the red region showing the cleavage site; panel c shows the detection of the induction activity of split-APOBEC1-BE 3.

In addition, the base editing system constructed by the application can keep Cas9 intact, so that the base editing system is compatible with other non-traditional base editors, such as CP-Cas9 derived base editors (Huang et al, 2019), internally mosaiced BE-PIGS base editing (Wang et al, 2019) and sgRNA framework modified sgBEs (Wang et al, 2020).

Claims

1. An inducible base editing system comprising a base editor in combination with rapamycin; the base editor comprises a deaminase;

the deaminase can be split based on any amino acid site; the rapamycin is combined with deaminase through the split amino acid site and induced to complete base editing.

2. The inducible base editing system of claim 1 wherein the FRB and FKBP of rapamycin are each coupled to a cleavage of a nonstructural loop of a deaminase amino acid cleavage site.

3. The inducible base editing system of claim 2 wherein the FRB and FKBP are fused to the N-and C-termini, respectively, of the deaminase amino acid cleavage site and dimerize to form a heterodimer, completing the assembly of the base editor.

4. The inducible base editing system of claim 1 wherein the deaminase split site is amino acid 44, 85, 118 or 147.

5. The inducible base editing system of claim 4 wherein the deaminase cleavage site is amino acid 85.

6. The inducible base editing system of any one of claims 1 to 5 wherein the deaminase is a cytosine deaminase.

7. The inducible base editing system of claim 6 wherein the cytosine deaminase is an APOBEC3A cytosine deaminase or APOBEC1 cytosine deaminase.

8. The inducible base editing system of claim 1, wherein the concentration of rapamycin is between 0.01 and 200 nM.

9. Use of the inducible base editing system of any one of claims 1 to 8 in gene editing.

10. A kit for gene editing comprising the inducible base editing system of any one of claims 1 to 9.