CN116064517A - Production mode of pilot editing gRNA and application thereof - Google Patents
Production mode of pilot editing gRNA and application thereof Download PDFInfo
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- CN116064517A CN116064517A CN202210904650.3A CN202210904650A CN116064517A CN 116064517 A CN116064517 A CN 116064517A CN 202210904650 A CN202210904650 A CN 202210904650A CN 116064517 A CN116064517 A CN 116064517A
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Abstract
The invention discloses a production mode of leader editing gRNA and application thereof, and provides a production mode of leader editing gRNA, wherein the production mode uses an RNA polymerase II promoter to transcribe pegRNA and a coding sgRNA required by leader editing. The invention uses RNA polymerase II promoter to replace traditional RNA polymerase III promoter to generate the pilot editing gRNA contained in the intron, thus obtaining a novel pilot editing tool p2PE3, and utilizing Doxycycline to control the generation of the pilot editing gRNA, thereby realizing controllable pilot editing, which is beneficial to further expanding the practicability of the pilot editing tool.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a generation mode of pilot editing gRNA and application thereof.
Background
The Prime Editing (PE) tool consists of Cas9 nickase and engineered reverse transcriptase, and corresponds to one pegRNA and one nicking sgRNA. Unlike normal sgrnas, the 3' end of pegRNA also carries a binding primer (Primer Binding Site, PBS) and a template (Reverse Transcription template, RTT) for repair. Reverse transcriptase reverse transcribes with PBS and RTT to obtain repaired DNA, which is used for directed mutation. By introducing a notch into the unedited strand of the nicking sgRNA, the editing efficiency of PE is greatly increased, and point mutation, small insertion or small deletion can be accurately introduced into the required site.
However, the pilot editing is not perfect, and the main limitation is its editing efficiency. Tests on human cell lines showed that the difference in editing efficiency of the lead editing in different cell lines was large and the difference in editing efficiency of different sites was also large. Liu et al and Nelson et al found that the 3' end of pegRNA greatly affected efficiency, mainly because the 3' end was easily degraded and complementary pairing with the 5' end was formed, affecting Cas9 binding. Liu et al, by adding a stem-loop structure of Csy4 recognition site at the 3' end of pegRNA, and linking with the linking sgRNA, cut by exogenously expressed Csy4 endonuclease, can greatly improve the efficiency of pilot editing, and the system is named ePE3.
However, the gRNA of ePE is produced by the RNA polymerase class III (Pol III) promoter, which results in the following problems with this system. First, pol III transcription is easily terminated by four to six consecutive U residues, so it is not suitable for editing sites on sgRNA containing poly (T); secondly, the Pol III promoter adds extra guanine or adenine at the 5 'end of the sgRNA, resulting in mismatch between the 5' end of the sgRNA and DNA, affecting the efficiency of high-fidelity SpCas9 variation; finally, the Pol III promoter is constitutively expressed and therefore cannot control the production of gRNA.
Therefore, we propose a generation mode of the lead editing gRNA and the application thereof.
Disclosure of Invention
The invention aims to solve the technical problems and provides a generation mode and application of a guide editing gRNA.
The technical scheme adopted by the invention is as follows:
the first aspect of the present invention provides a method for generating leader editing gRNA, which uses RNA polymerase II promoter to transcribe the pegRNA and the linking sgRNA required for the leader editing.
In a second aspect, the invention provides an isolated nucleic acid, a precursor RNA produced by the method of producing a leader edited gRNA of the first aspect of the invention, comprising:
(i) Two exons, a first and a second exon;
(ii) Editing required pegRNA and marking sgRNA in a pilot mode;
(iii) The three-segment Csy4 recognition site is divided into a first Csy4 recognition site, a second Csy4 recognition site and a third Csy4 recognition site;
(iv) A stretch comprising introns of (ii) and (iii);
the precursor RNA sequentially comprises the following components: the first exon, the first half of the intron sequence, the first Csy4 recognition site, the pegRNA, the second Csy4 recognition site, the linking sgRNA, the third Csy4 recognition site, the second half of the intron sequence, the second exon.
Further, the Csy4 endonuclease recognition sequence comprised by the precursor RNA comprises:
(a) A nucleotide sequence shown as SEQ ID NO. 1; or alternatively, the first and second heat exchangers may be,
(b) Has more than 95% identity with the nucleotide sequence shown in SEQ ID NO. 1 and maintains the function of being recognized by Csy4 endonuclease.
In a third aspect the invention provides a recombinant expression vector for a gRNA expressing an isolated nucleic acid as described in the second aspect of the invention.
A fourth aspect of the present invention provides a guided editing tool comprising:
(i) A fusion protein comprising a leader editor and an endonuclease;
(ii) An isolated nucleic acid according to the second aspect of the invention.
Further, the amino acid sequence of the lead editor is shown as SEQ ID NO. 2.
Further, the amino acid sequence of the endonuclease is shown as SEQ ID NO. 3.
Furthermore, the fusion protein uses a P2A fragment to connect a leader editor and endonuclease, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 4.
In a fifth aspect the invention provides an isolated encoding nucleic acid encoding a fusion protein in a guided editing tool according to the fourth aspect of the invention.
In a sixth aspect the invention provides an editor recombinant expression vector expressing an isolated nucleic acid encoding the fifth aspect of the invention.
In a seventh aspect, the invention provides an expression system comprising a gRNA recombinant expression vector according to the third aspect of the invention or an editor recombinant expression vector according to the sixth aspect of the invention, wherein the host cell of the expression system is a eukaryotic cell or a prokaryotic cell.
Further, the eukaryotic cell or prokaryotic cell is a eukaryotic cell of a human cell.
Further, the eukaryotic cells of the human cells are human embryonic kidney cells, human embryonic stem cells, human cervical cancer cells or human osteosarcoma cells.
Further, the human embryonic kidney cells, human embryonic stem cells, human cervical cancer cells or human osteosarcoma cells are HEK293T cells, H1 hESC cells, hela cells or U2OS cells.
In an eighth aspect, the invention provides the use of a leader editing gRNA according to the first aspect of the invention, an isolated nucleic acid according to the second aspect of the invention, a gRNA recombinant expression vector according to the third aspect of the invention, a leader editing tool according to the fourth aspect of the invention, an isolated encoding nucleic acid according to the fifth aspect of the invention, an editor recombinant expression vector according to the sixth aspect of the invention, an expression system according to the seventh aspect of the invention in eukaryotic gene editing.
Further, the uses include editing sites on pegRNA or a linking sgRNA that contain poly (T), enabling combinatorial editing, and enabling conditional or tissue specific editing.
The beneficial effects of the invention are as follows: the invention uses RNA polymerase II promoter to replace traditional RNA polymerase III promoter to generate leader editing gRNA contained in introns, thus obtaining a novel leader editing tool p2PE3. Compared with the traditional lead editing tool ePE3 based on RNA polymerase III promoter, p2PE3 has equivalent editing efficiency at the common site, but ePE3 has little effect when editing the site containing poly (T) on gRNA, but p2PE3 still has higher editing efficiency. Furthermore, when the gRNA is in the third position of the array, the editing efficiency of p2PE3 is significantly higher than ePE3 when the genes are simultaneously edited. The above results demonstrate that p2PE3 has great advantages when editing the sites where the gRNA contains poly (T). The advantage of p2PE3 is not only in editing efficiency, but also the traditional lead editing tool cannot realize editing controllability from the level of gRNA because RNA polymerase III promoter is constitutively expressed in cells. By using the inducible RNA polymerase II promoter, the p2PE3 can control the generation of the leader editing gRNA by utilizing Doxycycline, thereby realizing controllable leader editing, which is beneficial to further expanding the practicability of a leader editing tool.
Drawings
Fig. 1 is a schematic diagram of the pilot editing system p2PE3 of the present invention. The gRNA is transcribed and released in the form of an intron clomazone through a CAG promoter, and Csy4 endonuclease cuts a Csy4 recognition site sequence on the gRNA to generate functional pegRNA and a linking sgRNA, and the pegRNA and the PE2 protein play a role in leading editing together;
FIG. 2 is a schematic diagram of experimental results of example 3 of the present invention, wherein a is ePE3 and p2PE3 edit 6 endogenous gene loci on HEK293T cells; b is ePE and p2PE3 edits 6 endogenous gene loci on Hela cells; c is ePE and p2PE3 edits 6 endogenous gene loci on U2OS cells; d is the editing condition of the inducible p2PE3 on 6 endogenous gene loci on the H1 embryonic stem cells, and the editing condition is controlled by adding Doxycycline;
FIG. 3 is a schematic representation of the experimental results of example 4 of the present invention, showing the editing of sites containing poly (T) on 6 gRNAs on HEK293T cells by ePE and p2PE 3;
FIG. 4 is a schematic diagram of experimental results of example 5 of the present invention, wherein a is a schematic diagram of a gRNA array; b is the editing efficiency of ePE and p2PE3 on different array; c is ePE and p2PE3 index for different array; statistical analysis of editing efficiency of ePE and p2PE3 at different positions on array was performed, and normalized treatment was performed with the editing efficiency of ePE3 alone targeted as 1.
Detailed Description
The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to FIG. 1, a first aspect of the present invention provides a means of generating leader editing gRNA using RNA polymerase class II promoters to transcribe the desired pegRNA and the desired linking sgRNA for the leader editing.
In a second aspect, the invention provides an isolated nucleic acid, a precursor RNA produced by the method of producing a leader edited gRNA of the first aspect of the invention, comprising:
(i) Two exons, a first and a second exon;
(ii) Editing required pegRNA and marking sgRNA in a pilot mode;
(iii) The three-segment Csy4 recognition site is divided into a first Csy4 recognition site, a second Csy4 recognition site and a third Csy4 recognition site;
(iv) A stretch comprising introns of (ii) and (iii);
the precursor RNA sequentially comprises the following components: the first exon, the first half of the intron sequence, the first Csy4 recognition site, the pegRNA, the second Csy4 recognition site, the linking sgRNA, the third Csy4 recognition site, the second half of the intron sequence, the second exon.
The Csy4 endonuclease recognition sequence comprised by the precursor RNA comprises:
(a) A nucleotide sequence shown as SEQ ID NO. 1; or alternatively, the first and second heat exchangers may be,
(b) Has more than 95% identity with the nucleotide sequence shown in SEQ ID NO. 1 and maintains the function of being recognized by Csy4 endonuclease.
In a third aspect the invention provides a recombinant expression vector for a gRNA expressing an isolated nucleic acid as described in the second aspect of the invention.
A fourth aspect of the present invention provides a guided editing tool comprising:
(i) A fusion protein comprising a leader editor and an endonuclease;
(ii) An isolated nucleic acid according to the second aspect of the invention.
The amino acid sequence of the pilot editor is shown as SEQ ID NO. 2.
The amino acid sequence of the endonuclease is shown as SEQ ID NO. 3.
The fusion protein uses a P2A fragment to connect a leader editor and endonuclease, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 4.
In a fifth aspect the invention provides an isolated encoding nucleic acid encoding a fusion protein in a guided editing tool according to the fourth aspect of the invention.
In a sixth aspect the invention provides an editor recombinant expression vector expressing an isolated nucleic acid encoding the fifth aspect of the invention.
In a seventh aspect, the invention provides an expression system comprising a gRNA recombinant expression vector according to the third aspect of the invention or an editor recombinant expression vector according to the sixth aspect of the invention, wherein the host cell of the expression system is a eukaryotic cell or a prokaryotic cell.
The eukaryotic cell or prokaryotic cell is a eukaryotic cell of a human cell.
The eukaryotic cells of the human cells are human embryonic kidney cells, human embryonic stem cells, human cervical cancer cells or human osteosarcoma cells.
The human embryonic kidney cells, the human embryonic stem cells, the human cervical cancer cells or the human osteosarcoma cells are HEK293T cells, H1 hESC cells, heLa cells or U2OS cells.
In an eighth aspect, the invention provides the use of a leader editing gRNA according to the first aspect of the invention, an isolated nucleic acid according to the second aspect of the invention, a gRNA recombinant expression vector according to the third aspect of the invention, a leader editing tool according to the fourth aspect of the invention, an isolated encoding nucleic acid according to the fifth aspect of the invention, an editor recombinant expression vector according to the sixth aspect of the invention, an expression system according to the seventh aspect of the invention in eukaryotic gene editing.
The uses include editing sites on pegRNA or a linking sgRNA that contain poly (T), enabling combinatorial editing, and enabling conditional or tissue specific editing.
Example 1: precursor RNA construction in the lead editing system.
The precursor RNA expression plasmid pEF1a-PE2-Csy4 was constructed by P2A ligation using the one-step multipoint mutation kit (Vazyme, C215) from Nanjinouzan Biotechnology Co., ltd.) with EF1a promoter substituted for the CMV promoter in pCMV-PE2 (Addgene plasmid # 132775) and the DNA coding sequence of Csy4 endonuclease added to the C-terminus of PE2 protein. The sequences required for substitution and addition were synthesized by the company Kirschner Biotech Co. The complete plasmid sequence is shown as SEQ ID NO. 5, and the amino acid sequence of the expressed fusion protein is shown as SEQ ID NO. 6.
Example 2: and constructing a gRNA recombinant expression vector in the lead editing system.
The generation of the leader editing gRNA is based on an RNA polymerase II promoter, the promoters used by the recombinant expression vector are a CAG promoter and a TRE3GS promoter, and the corresponding leader editing systems are named as p2PE3 and i-p2PE3. While using a conventional leader editing system ePE3 based on an RNA polymerase class III promoter (U6 promoter) as a comparison.
The recombinant expression vector pGL3-CAG-mCherry (sgRNA intron) -hPDGK-EGFP producing gRNA was first constructed using the one-step multipoint mutation kit (Vazyme, C215) of Nanjinouzan Biotechnology Co.Ltd. Based on pGL3-U6-sgRNA-EGFP (Addgene plasmid # 107721) plasmid, the mCherry fragment containing sgRNA intron expressed by CAG promoter was synthesized by Kirsrui Biotech Co., ltd, and the U6-sgRNA in the vector was replaced. The complete plasmid sequence is shown in SEQ ID NO. 7. When constructing a recombinant expression vector pGL3-TRE3GS-mCherry (sgRNA intron) -hPDGK-EGFP capable of inducing gRNA, on the basis of pGL3-CAG-mCherry (sgRNA intron) -hPDGK-EGFP plasmid, the TRE3GS promoter is synthesized by the Kirschner Biotechnology Co., ltd., and the CAG promoter is replaced, and the complete plasmid sequence is shown as SEQ ID NO. 8.
The spacer region of pegRNA synthesizes forward and reverse oligonucleotides according to the designed sequence. The 3' extension sequence of pegRNA and the coding sgRNA sequence are connected through a Csy4 recognition site sequence and synthesized on an Oligo. The sgRNA scaffold is also added into the final vector through the synthesis of Oligo, the forward Oligo has the sequence shown in SEQ ID NO. 9, and the reverse Oligo has the sequence shown in SEQ ID NO. 10. Forward and reverse oligoas require the addition of an interface sequence based on the subsequently ligated vector. ePE3 and p2PE3 are different in the interface sequence of the forward Oligo of the spacer region of the pepRNA, ePE is ACCG and p2PE3 is GCAG. The Oligo is first annealed as shown in the following table:
TABLE 1 annealing System
| Component (A) | Dosage of |
| Forward Oligo (100. Mu.M) | 5μL |
| Reverse Oligo (100. Mu.M) | 5μL |
TABLE 2 annealing procedure
| Step (a) | Temperature (temperature) | | Cycle number | |
| 1 | 95 | 5min | 1 | |
| 2 | 95℃(-1℃/cycle) | |
10 | |
| 3 | 85℃(-0.1℃/cycle) | 2s | 600 | |
| 4 | 25℃(-2℃/cycle) | |
10 | |
| 5 | 4 | hold | 1 |
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 p2PE3 system links the annealed product to pGL3-CAG-mCherry (sgRNA intron) -hGGK-EGFP vector linearized by BsaI-HF; ePE3 the system ligates the annealed product into pGL3-U6-sgRNA-EGFP vector linearized by BsaI-HF; the i-PE3 system ligated the annealed product into pGL3-TRE3GS-mCherry (sgRNA intron) -hGGK-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+nick sgRNA annealing product (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: stably editing an endogenous gene locus in HEK293T cells, hela cells and U2OS cells by using p2PE 3; inducible editing was performed on human H1 embryonic stem cells using i-p2PE3 (Industle p2PE3, based on the TRE3GS promoter).
Step one: construction of gRNA plasmid
6 human endogenous genes were selected: RIT1, MSH2, CTLA4, FANCF, PDCD1 and RNF2, designing a pilot editing gRNA, wherein the used oligos are shown in the attached sequence table SEQ ID 11-40. Construction of the gRNA plasmid was performed as in example 2.
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). 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 1. Mu.g of pEF1a-PE2-Csy4 plasmid, and 0.5. Mu.g of gRNA 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 below:
TABLE 5 amplification System
| Component (A) | Dosage of |
| 2×Phanta Max Buffer | 25μL |
| dNTP Mix(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 6PCR 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/ |
||
| 8 | 72 | 3min | 1 | |
| 9 | 4 | 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 2×150bp 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.
Human H1 embryonic stem cells (purchased from WiCell Research Institute) were cultured on matrigel (STEMCELL Technologies) -coated dishes and cultured using mTeSR1 (STEMCELL Technologies) medium. The day before transfection, cells were dissociated with Accutase (Thermo), seeded at 50,000-75,000 cells/well into 24-well plates, and 1 μl of 10 μ M Y-27632 (Tocris) was added. Pre-transfection with Lipofectamine Stem reagent (Thermo) was used. The amount of plasmid transfected per well was 300ng of pEF1a-PE2-Csy4 plasmid, 300ng of gRNA plasmid, pEF1a-SV40LT-hMLH1, respectively NTD -NLS plasmid 200ng, pEF1a-TetOn-3G plasmid 200ng. The induction group was simultaneously supplemented with 1. Mu.g/ml Doxycycline. Editing efficiency was identified 48 hours after transfection as above.
Step three: analysis of results
The 6 sites were edited by p2PE3 on HEK293T cells, heLa cells, U2OS cells as shown in FIGS. 2 (a) -2 (c). p2PE3 was comparable to ePE in efficiency in HEK293T cells and U2OS cells, and slightly lower than ePE in Hela cells. FIG. 2 (d) shows that p2PE3 was inducible for 6 sites in human H1 embryonic stem cells, and was not able to be efficiently edited only in the presence of Doxycycline (+Dox) but not in the absence of Doxycycline (-Dox).
Example 4: efficient editing of poly (T) -containing sites in gRNA in HEK293T cells was performed using p2PE3.
Step one: construction of gRNA plasmid
6 human endogenous genes were selected: BRCA1, COL1A2, GPSM2, ADGRV1, OL9A1, TSC1, lead edit gRNA was designed, all with poly (T) on them. The oligos used are shown in the attached sequence table SEQ ID Nos. 41-70. Construction of the gRNA 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. 3. The p2PE3 based on the RNA polymerase II promoter can efficiently edit the site containing poly (T) of the gRNA, and ePE based on the RNA polymerase III promoter has poor editing effect on the site containing poly (T) of the gRNA. It is demonstrated that p2PE3 has great advantages in editing sites where gRNA contains poly (T).
Example 5: the simultaneous editing of 3 endogenous genes was performed in HEK293T cells using p2PE3.
Step one: construction of sgRNA plasmids
3 human endogenous genes were selected: MKL2, MYH7 and NKX2-5, the pilot editing gRNA was designed. The oligos used are shown in the attached sequence table SEQ ID Nos. 71-85. Construction of the gRNA plasmid was performed as in example 2. Furthermore, the gRNAs were ligated using Csy4 recognition site sequences in the order of FIG. 4 (a), resulting in 3 gRNA arrays. The gRNA array was synthesized by Kirschner Biotechnology Co., ltd and cloned into pGL3-CAG-mCherry (sgRNA intron) -hGGK-EGFP vector or pGL3-U6-sgRNA-EGFP vector. The sequence of the gRNA array is shown in the attached sequence table SEQ ID 86-88.
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. 4. It can be seen that when gRNA was edited alone, or in the first place in Array, the editing efficiency of p2PE3 and ePE3 was comparable; when the gRNA is positioned at the second position of the Array, the editing efficiency of the p2PE3 is slightly higher than ePE3; when the gRNA is in the third position of Array, the editing efficiency of p2PE3 is significantly higher than ePE3. Illustrating that p2PE3 has great advantages when performing combinatorial editing.
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 (16)
1. A method for producing leader editing gRNA, characterized in that the pegRNA and the ringing gRNA required for leader editing are transcribed using an RNA polymerase II promoter.
2. An isolated nucleic acid, wherein the precursor RNA produced by the method of producing a leader edited gRNA of claim 1, comprises:
(i) Two exons, a first and a second exon;
(ii) Editing required pegRNA and marking sgRNA in a pilot mode;
(iii) The three-segment Csy4 recognition site is divided into a first Csy4 recognition site, a second Csy4 recognition site and a third Csy4 recognition site;
(iv) A stretch comprising introns of (ii) and (iii);
the precursor RNA sequentially comprises the following components: the first exon, the first half of the intron sequence, the first Csy4 recognition site, the pegRNA, the second Csy4 recognition site, the linking sgRNA, the third Csy4 recognition site, the second half of the intron sequence, the second exon.
3. An isolated nucleic acid according to claim 2, wherein the precursor RNA comprises a Csy4 endonuclease recognition sequence comprising:
(a) A nucleotide sequence shown as SEQ ID NO. 1; or alternatively, the first and second heat exchangers may be,
(b) Has more than 95% identity with the nucleotide sequence shown in SEQ ID NO. 1 and maintains the function of being recognized by Csy4 endonuclease.
4. A gRNA recombinant expression vector, wherein an isolated nucleic acid of claim 2 is expressed.
5. A guided editing tool comprising:
(i) A fusion protein comprising a leader editor and an endonuclease;
(ii) An isolated nucleic acid according to claim 2.
6. The guided editing tool of claim 5, wherein the leader editor has an amino acid sequence as set forth in SEQ ID No. 2.
7. A guided editing tool according to claim 5, wherein the endonuclease has an amino acid sequence as shown in SEQ ID NO. 3.
8. The guided editing tool of claim 5, wherein the fusion protein uses a P2A fragment to link a leader editor to an endonuclease having an amino acid sequence as set forth in SEQ ID NO. 4.
9. An isolated coding nucleic acid encoding a fusion protein of a guided editing tool of claim 5.
10. An editor recombinant expression vector expressing an isolated nucleic acid encoding the nucleic acid of claim 9.
11. An expression system comprising a gRNA recombinant expression vector of claim 4 or an editor recombinant expression vector of claim 10, wherein the host cell of the expression system is a eukaryotic cell or a prokaryotic cell.
12. An expression system according to claim 11, wherein the eukaryotic or prokaryotic cell is a eukaryotic cell of a human cell.
13. An expression system according to claim 12, wherein the eukaryotic cells of said human cells are human embryonic kidney cells, human embryonic stem cells, human cervical cancer cells or human osteosarcoma cells.
14. An expression system according to claim 13, wherein the human embryonic kidney cells, human embryonic stem cells, human cervical cancer cells or human osteosarcoma cells are HEK293T cells, H1 hESC cells, hela cells or U2OS cells.
15. Use of a lead-edited gRNA of claim 1, an isolated nucleic acid of any one of claims 2-3, a gRNA recombinant expression vector of claim 4, a lead-editing tool of any one of claims 5-8, an isolated encoding nucleic acid of claim 9, an editor recombinant expression vector of claim 10, an expression system of any one of claims 11-14 in eukaryotic gene editing.
16. The use according to claim 15, comprising editing a poly (T) -containing site on a pegRNA or a nicking sgRNA, effecting a combinatorial editing, and effecting a conditional or tissue specific editing.
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