CN116396952A - Pilot editing system and gene editing method - Google Patents
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Abstract
The invention discloses a pilot editing system and a gene editing method. The leader editing system comprises a leader editor with nuclease activity, pegRNA comprising a template of homologous sequence, and further comprises a protein with non-homologous end repair inhibitory properties. According to the invention, a non-homologous tail end repair inhibitory protein is introduced on the basis of a nuclease leading editor, so that the purity of an editing product is obviously reduced and improved by non-precise editing; meanwhile, the length of the homologous sequence depended on restoration is optimized, and the optimal length, namely the length of the homologous sequence of 20bp, is found through efficiency comparison; the combination of the two optimization modes further improves the accurate editing efficiency. Based on the repair mode of homology dependence, the uPEn can realize efficient small fragment insertion, deletion and replacement editing, and can also realize efficient editing at sites which are difficult to edit by the traditional lead editor, which is beneficial to further expanding the practicability of the lead editing tool.
Description
Technical Field
The invention belongs to the technical field of gene editing, and particularly relates to a pilot editing system and a gene editing method.
Background
At present, a series of gene editing tools based on CRISPR-Cas system derivation bring great hope for realizing accurate editing of genome. The recent advent of Prime Editing (PE) represents a breakthrough in a very advanced gene Editing technology that can introduce various types of base mutations and small fragment insertions, deletions and substitutions into the genome while avoiding double strand breaks in DNA (Double Stand Break, DSB).
The leader editing system consists of a PE protein expressed by fusion of Cas9 nickase and reverse transcriptase and a prime editing guide RNA (pegRNA) with template and leader, wherein the pegRNA consists of 3 parts including single-guide RNA (sgRNA), primer binding site (Prime Binding Site, PBS) and reverse transcription template with editing information (Reverse Transcription template, RT template). The editing efficiency of the first generation PE1 and the second generation PE2 at the target site is limited, and finally, researchers construct PE3/PE3b, and the editing efficiency is further improved by coexpression of a nick-sgRNA targeting a complementary DNA single strand and causing a nick, and by utilizing a cell endogenous repair mechanism, the proportion of repair with a reverse transcription strand as a template is improved.
Compared with the previous gene editing system, the pilot editing function is stronger, the safety is higher, and the method has been widely applied to genome editing of rice, wheat, zebra fish, mouse embryo and other species. It is worth noting that one important issue with lead editing systems is the limited editing efficiency. Especially, when inserting, deleting and replacing editing is carried out on sequences with the length of tens of bp, the editing efficiency is very low, and the application of the PE3 system is further limited because two gRNAs are required to act together to realize effective editing.
Recently, a series of work optimized for PE systems has improved editing efficiency to some extent, for example, anzalone et al (Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing Nature Biotechnology, doi.org/10.1038/s 41587-021-01133-w) uses a strategy of double pegRNA, improving efficiency of small fragment editing, but greatly increasing difficulty in PE system design and complexity of the system itself, increasing difficulty in application thereof. Another study reported that constructing a lead editing system with nuclease active Cas9 by Adikusuma et al (Optimized nickase-and nucleic-based prime editing in human and mouse cells, nucleic Acids Research, doi.org/10.1093/nar/gkab 792) is free of dependency on nick-sgRNA, but results in a large amount of non-precise editing, and the precise editing efficiency is low.
Nevertheless, editing efficiency for PE is still suboptimal at most of the genomic sites tested, and editing of small fragments is still a major difficulty for PE editing. In addition, the dependence of the PE system on two gRNAs was not improved.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides development and application of a pilot editing system.
A leader editing system comprising a leader editor having nuclease activity, a pegRNA comprising a template of homologous sequence, and a protein having non-homologous end repair inhibitory properties. According to the invention, by introducing the protein with non-homologous end repair inhibition, non-precise editing can be remarkably reduced and the purity of an editing product can be improved by inhibiting non-homologous end repair.
Preferably, the non-homologous end repair inhibitory protein is a protein capable of specifically targeting the 53BP1 protein and inhibiting 53BP1 protein activity.
Further preferred, the non-homologous end repair inhibitory protein is a ubiquitinated variant G08 or a mutant of ubiquitinated variant G08 having an I44A mutation, wherein the amino acid sequence of ubiquitinated variant G08 is as set forth in SEQ ID NO: 1.
Preferably, the leader editor with nuclease activity comprises a fusion expressed nuclease active Cas9 protein and a reverse transcriptase protein. Further preferably, the amino acid sequence of the leader editor with nuclease activity is as shown in SEQ ID NO: 2.
Preferably, the pegRNA containing the homologous sequence template comprises a reverse transcription template with editing information, and the sequence length of the reverse transcription template homologous to the genome sequence in the gene editing object is 15-25 bp. Most preferably 20bp. The invention optimizes the length of the homologous sequence depending on repair, and the efficiency comparison shows that the effect is better at 15-25 bp, and the optimal length is 20bp.
The invention also provides a recombinant expression sequence for leading editing, which is used for expressing the leading editing system, and the recombinant expression system comprises:
(1) A first gene sequence for expressing a leader editor having nuclease activity,
(2) A second gene sequence for expressing a protein having non-homologous end repair inhibitory properties,
(3) A third gene sequence for expressing a pegRNA comprising a homologous sequence template.
Preferably, the first gene sequence and the second gene sequence are expressed using the same expression vector, and the first gene sequence and the second gene sequence are linked by a self-cleaving short peptide encoding gene sequence, such that the leader editor having nuclease activity and the protein having non-homologous end repair inhibitory properties are fusion expressed by the self-cleaving short peptide. The self-cleaving short peptide will automatically cleave upon expression of the fusion protein, becoming two proteins. The two proteins were separately de-expressed, requiring the addition of transfection with one expression vector, and no difference was seen in the results. The advantage is that there is one less expression vector, which is more compact and convenient
The invention also provides a gene editing method, which uses the recombinant expression sequence to design pegRNA according to the sequence to be subjected to gene editing in the gene editing object to obtain a third gene sequence, and the first gene sequence, the second gene sequence and the third gene sequence are all introduced into the gene editing object to carry out gene editing to obtain a product after gene editing.
The types of gene editing of the invention can be various base editing, small fragment insertion, deletion, substitution editing, combined editing and conditional or tissue specific editing.
The gene editing object is eukaryotic cells or prokaryotic cells. Such as human cells or other mammalian cells. Preferably, the gene editing object is a human embryonic kidney cell, a human cervical cancer cell or a human osteosarcoma cell, such as a HEK293T cell, a Hela cell or a U2OS cell.
The beneficial effects of the invention are as follows: the present invention uses a nuclease leader editor to bind the ubiquitinated variants and by optimizing the pegRNA a new leader editing tool uPEn is obtained that is more efficient and requires only one pegRNA. Compared with the traditional lead editing tool, the lead editor with nuclease activity does not need additional nick-sgRNA to generate a notch to improve editing efficiency, so that a PE system becomes simpler, meanwhile, double-strand cutting and single-strand cutting are activated in a different repair mode, the inhibition of MMR on the traditional PE can be eliminated, and the total editing efficiency is increased but the editing purity is reduced. Therefore, we introduced a non-homologous end repair inhibitory protein-ubiquitinated variant based on the nuclease leader editor by inhibiting non-homologous end repair. The result shows that the addition of the ubiquitinated variant obviously reduces the inaccurate editing and improves the purity of the editing product; meanwhile, the length of the homologous sequence depended on restoration is optimized, and the optimal length, namely the length of the homologous sequence of 20bp, is found through efficiency comparison; the combination of the two optimization modes further improves the accurate editing efficiency. Based on the repair mode of homology dependence, the uPEn can realize efficient small fragment insertion, deletion and replacement editing, and can also realize efficient editing at sites which are difficult to edit by the traditional lead editor, which is beneficial to further expanding the practicability of the lead editing tool.
Drawings
FIG. 1 is a schematic diagram of the principle of gene editing in the pilot editing system of the present invention.
Fig. 2 is a schematic diagram of experimental results of example 3 of the present invention, showing the editing situation of the uPEn system compared with the PEn.
FIG. 3 is a schematic diagram of experimental results of embodiment 4 of the present invention, wherein A is the influence of the homologous sequence length on the efficiency of uPEn editing; b is the change trend of editing efficiency with the increase of the length of the homologous sequence.
FIG. 4 is a schematic illustration of the design of uPEn mediated insert, delete and replace editing in example 5 of the present invention.
FIG. 5 is a graph showing the comparison of editing efficiency of uPEn and PE2max, PE3max, PE5max at four endogenous gene loci in example 5 of the present invention, wherein A to D are CDKL5, CXCR4, DNMT1 and RNF2, respectively.
FIG. 6 is a graph showing the results of statistical analysis of editing efficiencies of uPEn and PE2max, PE3max, and PE5max at all sites in example 5 according to the present invention.
FIG. 7 is a schematic diagram of experimental results of example 6 of the present invention, wherein A is the comparison of editing efficiency of uPEn and PE3max at 10 endogenous gene loci of 293T cells; b is the comparison of the editing efficiency of uPEn and PE5max on 6 endogenous gene loci of HeLa cells; c is the comparison of the editing efficiency of uPEn and PE5max at 6 endogenous gene loci of U2OS cells.
Detailed Description
Referring to FIG. 1, the present invention provides a leader editing system comprising a leader editor having nuclease activity, and a protein having non-homologous end repair inhibitory properties, and a pegRNA comprising a homologous sequence template. The design principle of the optimized pegRNA is shown in FIG. 1.
Example 1: expression vector construction of uPEn fusion protein in lead editing system of the invention
The ubiquitination variant sequence is optimized and synthesized by Jin Weizhi biotechnology Co-Ltd, and is formed by connecting DNA coding sequences of a single-step multipoint mutation kit (Vazyme, C215) of Nanjinovirzan biotechnology Co-Ltd and a ubiquitination variant (UbvG 08-I44A, the amino acid sequence of which is shown as SEQ ID NO:1, and the UbvG08-I44A is UbvG08, and the I44A point mutation is added on the basis of the UbvG 08) through P2A (the amino acid sequence of which is GSGATNFSLLKQAGDVEENPGP) at the C end of PEmax protein in pCMV-PEmax (Addgene plasmid # 174820). The required sequences for substitution and addition were synthesized by Jin Weizhi Biotech Co. The complete plasmid sequence is shown in SEQ ID NO:4, the amino acid sequence of the expressed fusion protein is shown as SEQ ID NO: 3. The expressed fusion protein is a leader editor-P2A-ubiquitinated variant, wherein the leader editor (amino acid sequence shown in SEQ ID NO: 2) comprises a Cas9 protein and a reverse transcriptase protein which fuse expressed nuclease activity, SEQ ID NO:3 is a variant with an I44A mutation.
Example 2: construction of pegRNA recombinant expression vector in lead editing system
The invention discloses a method for constructing a recombinant expression vector for generating pegRNA by using a one-step multipoint mutation kit (Vazyme, C215) of Nanjinozan biotechnology limited company on the basis of pGL3-U6-sgRNA-EGFP (Addgene plasmid # 107721) plasmid, wherein the spacer region and the 3' extension sequence of the pegRNA synthesize forward and reverse oligonucleotide sequences (Oligo) according to designed sequences. sgRNA scaffold was also added to the final vector by synthesis of Oligo, the forward Oligo sequence as set forth in SEQ ID NO:5, the sequence of the reverse Oligo is shown as SEQ ID NO: shown at 6. Forward and reverse oligoas require the addition of an interface sequence based on the subsequently ligated vector. The Oligo was first annealed and the annealing system and annealing procedure are shown in tables 1 and 2.
TABLE 1 annealing System
| Component (A) | Dosage of |
| Forward Oligo (100. Mu.M) | 5μL |
| Reverse Oligo (100. Mu.M) | 5μL |
TABLE 2 annealing procedure
After the sgRNA scaffold annealing, 5' phosphorylation was added, the reaction system was as shown in table 3 below, and the reaction conditions were 37 ℃ for 1h incubation.
TABLE 3 reaction system
| Component (A) | Dosage of |
| 10×T4 PNK Reaction Buffer | 10μL |
| T4 PNK | 1μL |
| 10mM ATP | 2μL |
| sgRNA scaffold annealed product | 10μL |
| dd H 2 O | Up to 100 mu L |
The annealed product was ligated into pGL3-U6-sgRNA-EGFP vector linearized by BsaI-HF. The system is shown in Table 4 below and incubated at 16℃for 1h.
Table 4 System
| Component (A) | Dosage of |
| Solution I | 5μL |
| sgRNA scaffold 5' phosphorylation products | 2μL |
| Spacer annealing products (10. Mu.M) | 1μL |
| 3' extension annealing products (10. Mu.M) | 1μL |
| Linearization carrier | 1μL |
All recombinant and ligation products were transformed using chemically competent cells. The method comprises the following steps: the product was added to DH 5. Alpha. Competent cells thawed on ice and incubated on ice for 30min. After heat shock at 42℃for 90s, the cells were returned to ice and incubated for 2min. Subsequently, 200. Mu.L of liquid LB medium was added and the mixture was shaken in a shaker at 37℃for 30min. The bacterial liquid was then spread evenly on LB solid medium containing ampicillin, and cultured in an incubator at 37℃for 14 hours. The monoclonal was picked for Sanger sequencing validation. Positive clones were cultured with LB liquid medium containing ampicillin at 37℃for 12-14h, and plasmids were extracted with plasmid DNA miniprep kit or endotoxin-free plasmid miniprep kit.
Example 3: stable insertion editing of endogenous gene loci in HEK293T cells using uPEn
Step one: construction of pegRNA plasmid
4 human endogenous genes were selected: FANCF, UBE3A, SHANK3-1 and SHANK3-2, designing and leading editing pegRNA, wherein the used oligos are shown in a sequence table SEQ ID NO:7-22. Construction of the pegRNA plasmid was performed as in example 2. The constructed pegRNA sequence is a spacer sequence, a scaffold structural sequence and a 3' extension sequence (RT sequence) from the 5' end to the 3' end.
Step two: cell culture transfection and identification
HEK293T cells (purchased from ATCC) were inoculated in DMEM high sugar broth (HyClone, SH30022.01B) supplemented with 10% fbs, which contained 1%Penicillin Streptomycin (v/v) (Gibco). Cells were seeded into 24-well plates the day prior to transfection to give a cell concentration of around 70% on the day of transfection. The amount of plasmid transfected per well was 0.9. Mu.g for uPEn plasmid and 0.3. Mu.g for pegRNA plasmid, respectively. The plasmid was mixed in 50. Mu.l of Opti-MEM (Gibco, 11058021) medium. Mu.l of Lipofectamine2000 transfection reagent (Thermo, 11668019) was mixed into 50. Mu.l of Opti-MEM medium and mixed well and allowed to stand for 5 minutes. The Opti-MEM mixed with the plasmid was added to the Opti-MEM mixed with Lipofectamine2000, and the mixture was stirred and stirred at a slow speed and allowed to stand for 20 minutes. Opti-MEM mixed with plasmid and Lipofectamine2000 was added to each 24-well plate. DMEM with 10% fbs was used 6 hours after transfection. Cells were resuspended after pancreatin digestion with medium containing 10% fbs 72 hours after transfection, and EGFP positive cells were collected by BD FACS AriaIII sorting after sieving through 40 μm filter.
The harvested cells were lysed with a DNA flash extract at 68℃for 20min and then subjected to inactivation at 98℃for 2min. The vicinity of the target site is amplified with a DNA high-fidelity polymerase. The amplification system is shown in Table 5 and the amplification procedure is shown in Table 6.
TABLE 5 amplification System
| Component (A) | Dosage of |
| 2×Phanta Max Buffer | 25μL |
| dNTPMix(10mM) | 1μL |
| Phanta Max Super-Fidelity DNA Polymerase | 1μL |
| Forward primer/reverse primer (10. Mu.M) | 1/1μL |
| DNA template | 1μL |
| dd H 2 O | Is added to 50 mu L |
TABLE 6 PCR procedure
| Step (a) | Temperature (temperature) | | Cycle number | |
| 1 | 95 | 3min | 1 | |
| 2 | 98 | 10s | ||
| 3 | 68℃(-1℃/cycle) | |
10 | |
| 4 | 72℃ | 30s/ |
||
| 5 | 98℃ | 10s | ||
| 6 | 58 | 20s | 25 | |
| 7 | 72℃ | 30s/kb | ||
| 8 | 72 | 3min | 1 | |
| 9 | 4℃ | Hold (hold) | 1 |
The amplified product was purified using a PCR clean kit. Editing efficiency was accurately assessed by second generation high throughput sequencing. The PCR products to be sequenced are sent to Northlasiogenic biotechnology Co., ltd or Annoeuda Gene technology Co., ltd for amplicon pool-building sequencing. After cleardata is obtained, read lengths of 2×250bp are spliced using an AdapterRemoval, then all processed read lengths are aligned to the target sequence using the bwa mem algorithm, and the results are ranked using samtools alignment. Finally, the python program is written to calculate the editing efficiency and indel.
Step three: analysis of results
The editing of 4 sites by uPEn on HEK293T cells is shown in figure 2. The editing efficiency of uPEn on HEK293T cells is far higher than that of PEn, and the level of doubling indels is also obviously reduced, which proves that the editing efficiency is obviously improved by inhibiting non-homologous end repair through ubiquitinated variants, and meanwhile, the dependence on nick-sgRNA is also eliminated.
Example 4: the optimized pegRNA improves the editing efficiency
Step one: construction of pegRNA plasmid
3 human endogenous genes were selected: EMX1, FAM17A, PRNP, designed and edited pegRNA, these pegRNA contains 0bp, 5bp, 10bp, 15bp, 20bp, 25bp homologous sequences respectively. The oligos used are shown in the sequence table SEQ ID NO:23-64. Construction of the pegRNA plasmid was performed as in example 2.
Step two: cell culture transfection and identification
HEK293T cells (purchased from ATCC) were cultured, transfected and identified as per step two in example 3.
Step three: analysis of results
The correlation results are shown in fig. 3A and 3B. On all three editing sites, with the continuous increase of the length of the homologous region, the editing efficiency of uPEn is also continuously increased, when the homologous sequence reaches 15bp, the editing efficiency reaches the maximum value under the three lengths of HR15, HR20 and HR25, and the tendency of the editing efficiency is not increased, and by comprehensive comparison, we determine that the pegRNA containing the length of the homologous sequence of 20bp is the optimal pegRNA of the uPEn system, and then the pegRNA is the optimal design principle of the pegRNA.
Example 5: small fragment insertion, deletion and substitution editing using uPEn in HEK293T cells
Step one: construction of pegRNA and nick-sgRNA plasmids
4 human endogenous genes were selected: CDKL5, CXCR4, RNF2 and DNMT1, pilot editing pegRNA was designed. The oligos used are shown in the sequence table SEQ ID NO:65-96. Construction of the gRNA plasmid was performed as in example 2. Furthermore, pegRNA and corresponding nick-sgRNA mediating insertion, deletion and substitution were designed according to the design principle of FIG. 4, respectively. The oligos used by the nick-sgRNA are shown in the sequence listing SEQ ID NO:97-104. The Oligo was first annealed and the annealed product was ligated into pGL3-U6-sgRNA-mCherry vector linearized with BsaI-HF. The system is shown in Table 5 below and incubated at 16℃for 1h.
Table 5 System
| Component (A) | Dosage of |
| Solution I | 3μL |
| Spacer annealing products (10. Mu.M) | 2μL |
| Linearization carrier | 1μL |
Step two: cell culture transfection and identification
HEK293T cells (purchased from ATCC) were cultured, transfected and identified as per step two in example 3.
Step three: analysis of results
The relevant results are shown in fig. 5 and 6, and the editing efficiency of the uPEn-mediated small fragments is obviously higher than that of the traditional PE2max, PE3max and PE5max, which illustrate the great advantage of uPEn in editing the small fragments.
Example 6: base mutation editing in HEK293T cells, U2OS cells and HeLa cells using uPEn
Step one: construction of pegRNA and nick-sgRNA plasmids
10 human endogenous genes were selected: ALDOB, BCL11A, CCR, DNMT1, EGFR, EMX1, KCNA1, MECP2, RIT1, VISTA1, design leader editing pegRNA. The oligos used are shown in the sequence table SEQ ID NO:105-144. Construction of the pegRNA plasmid was performed as in example 2. In addition, the corresponding nick-sgRNA was constructed in the manner of example 5. The oligos sequence listing SEQ ID NO used by the nick-sgRNA: 145-164.
Step two: cell culture transfection and identification
HEK293T cells, hela cells, U2OS cells (purchased from ATCC) were inoculated in DMEM high sugar broth (HyClone, SH30022.01B) supplemented with 10% fbs, which contained 1%Penicillin Streptomycin (v/v) (Gibco). Transfection and identification was performed as in step two of example 3.
Step three: analysis of results
The correlation results are shown in fig. 7. It can be seen that uPEn can be effectively edited in various cell lines, and at some sites where conventional PE3max or PE5max cannot be edited, uPEn system still shows higher editing efficiency, indicating the great advantage of uPEn for some sites where editing is difficult.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A leader editing system comprising a leader editor having nuclease activity, a pegRNA comprising a template of homologous sequence, and a protein having non-homologous end repair inhibitory properties.
2. The lead editing system of claim 1, wherein the non-homologous end repair inhibitory protein is a protein capable of specifically targeting 53BP1 protein and inhibiting 53BP1 protein activity.
3. The lead editing system of claim 2, wherein the non-homologous end repair inhibitory protein is a ubiquitinated variant G08 or a mutant of ubiquitinated variant G08 having an I44A mutation, wherein the amino acid sequence of ubiquitinated variant G08 is as set forth in SEQ ID NO: 1.
4. The lead editing system of claim 1, wherein the lead editor having nuclease activity comprises a fusion expressed nuclease-active Cas9 protein and a reverse transcriptase protein.
5. The leader editing system according to claim 4, wherein the leader editor having nuclease activity has an amino acid sequence as set forth in SEQ ID NO: 2.
6. The leader editing system according to claim 1, wherein the pegRNA comprising the homologous sequence template comprises a reverse transcription template with editing information, wherein the sequence length homologous to the genomic sequence in the gene editing object in the reverse transcription template is 15 to 25bp.
7. A recombinant expression sequence for leader editing for expressing the leader editing system according to any one of claims 1 to 6, comprising:
(1) A first gene sequence for expressing a leader editor having nuclease activity,
(2) A second gene sequence for expressing a protein having non-homologous end repair inhibitory properties,
(3) A third gene sequence for expressing a pegRNA comprising a homologous sequence template.
8. The recombinant expression sequence for leader editing according to claim 7, wherein the first gene sequence and the second gene sequence are expressed using the same expression vector, and wherein the first gene sequence and the second gene sequence are linked by a self-cleaving short peptide encoding gene sequence, whereby the leader editor having nuclease activity and the protein having non-homologous end repair inhibitory properties are fusion expressed by the self-cleaving short peptide.
9. A gene editing method is characterized in that the recombinant expression sequence of claim 7 is used, pegRNA is designed according to a sequence to be subjected to gene editing in a gene editing object to obtain a third gene sequence, and the first gene sequence, the second gene sequence and the third gene sequence are all introduced into the gene editing object to carry out gene editing to obtain a product after gene editing.
10. The method of claim 9, wherein the gene editing object is a eukaryotic cell or a prokaryotic cell.
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