Gene knockout method based on base editing and its application

Abstract

Gene knockout method based on base editing and its application is provided. The gene knockout method comprises: selecting a 20 bp-NGG target sequence of the coding region of the gene to be knocked out, so that it contains a complete target codon CAA, CAG or CGA; and using sgRNA sequence to locate BE3 to the target sequence, to convert the target single-base C of the target codon into T and thus introduce a corresponding termination codon TAA, TAG orTGA in order to realize the knockout, wherein the target single-base C is located preferably on site 4-8 in the target sequence, the interval between the target codon and NGG is 12 to 14 bp, and the upstream base (H) near the target codon cannot be G; and the sgRNA sequence is a 20 bp sequence complementary to the target sequence.

Claims

1. An ex vivo method of knocking out a human PD1 gene comprising: selecting a 20 bp-NGG target sequence of the coding region of the gene to be knocked out, such that it contains a complete target codon CAA, CAG or CGA; and using sgRNA sequence to position BE3 Base Editor 3 (BE3) in the target sequence to convert the target single-base C of the target codon into T, in order to introduce a corresponding termination codon TAA, TAG or TGA for realization of the gene knockout, wherein the target single-base C is located between site 1 to 8 of the target sequence; the interval of the target codon and NGG is 12 to 14 bp; the upstream base adjacent the target codon cannot be G; wherein the human PD1 gene is in a human T cell line, and the sgRNA sequences are complementary to the 20 bp-NGG target sequences and are selected from the group consisting of “cagcaaccag acggacaage tgg” as identified by SEQ ID NO:6 and “cggccagttc caaaccctgg tgg” as identified by SEQ ID NO:7; and wherein BE3 is rAPOBEC 1-SaCas9-NLS-UGI-NLS as identified by the nucleotide of SEQ ID NO:1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustratively shows knocking out the target gene by CT mutation according to the present invention; and

(2) FIGS. 2-5 illustratively show different BE3 structures.

EMBODIMENTS

(3) Firstly, different BE3 was constructed, as shown in FIGS. 2-5. After the fusion of different Cas9 nickase and cytosine deaminase (APOBEC1), the following four BE3 were formed: (1) rapobecl-sacas9-nls-ugi-nls, FIG. 2, SEQ ID NO:1;

(4) (2) 3 xugi-rAPOBEC1-SaCas9-NLS—the UGI-NLS, FIG. 3, SEQ ID NO:2;

(5) (3) rAPOBEC1SpCas9-NLS—the UGI-NLS, FIG. 4, SEQ ID NO:3;

(6) (4) 3 xugi-rAPOBEC1-SpCas9-NLS—the UGI-NLS, FIG. 5, SEQ ID NO:4.

(7) In the following gene knockout, any of the above BE3 can be used, preferably (3) or (4).

(8) Next, in the design of sgRNA, the base point editing is to use sgRNA to locate BE3 or target it to the specific sites. The key of the invention is the selection and design of target gene specific sgRNA. The present invention selects and designs sgRNA as below:

(9) selecting a 20 bp-NGG target sequence (PAM sequence) of the coding region of the gene to be knocked out, such that it includes a complete target codon CAA, CAG or CGA;

(10) the target single base C is preferably located on site 4-8 (in the left end) of the target sequence, the interval between the target codon and NGG is preferably 14 bp, and the upstream base (H) near the target codon cannot be G;

(11) preparing a sequence of 20 bp sgRNA complementary to the target sequence.

(12) Alternatively, a CCN-20 bp target sequence (PAM) of the coding region of the gene to be knocked out is selected to include a complete target codon TGG, and the downstream base (D) near the target codon cannot be C. Accordingly, the target single base G is preferably located on site 4-8 (at the right end) of the target sequence, and the interval of the target codon and CCN is preferably 14 bp. With respct to the 8 different target genes (human PD1, LAG3, TIGIT, VISTA, 2B4 and CD160, and mouse TIM3 and LAG3), the following target gene sequences are selected to design the corresponding sgRNA according to the present invention (the bold and underlined portions represent PAMs; and the italic and underlined portions represent the candidate mutation codes):

(13) TABLE-US-00001 1. hPD-1 Sg-1: CTACAACTGGGCTGGCGGCCAGG Sg-2: CAGCAACCAGACGGACAAGCTGG Sg-3: CGGCCAGTTCCAAACCCTGGTGG 2. hLAG3 Sg-1: CCAGACCATAGGAGAGATGTGGG Sg-2: CCATAGGAGAGATGTGGGAGGCT Sg-3: CCGGCGGCGCCCTCCTCCTGGGG 3. hTIGIT Sg-1: GATCGAGTGGCCCCAGGTCCCGG 4. hVISTA Sg-1: CCTTCTACAAGACGTGGTACCGC 5. 2B4 Sg-1: GCAGCTCAGCAGCAGGACAGTGG 6. hCD160 Sg-1: AAAACAGCTGAGACTTAAAAGGG 7. mTIM3 Sg-1: CCTCGTGCCCGTCTGCTGGGGCA 8. mLAG3 Sg-1: CCAGACCATAGGAGAGATGTGG

(14) For the above-selected target gene sequences, human PD1 (3), LAG3 (3), TIGIT, VISTA, 2B4 and CD160, and mouse TIM3, LAG3, corresponding sgRNA expression vectors are built and different sgRNAs are imported into pGL3-U6-sgRNA respectively.

Example 1

(15) In the cell line, BE3 mediated base editing is performed, and the termination codon was introduced to realize gene knockout. The knockout of the cell lines is operated regularly (through electrotransfection or liposome transfection), taking liposome transfection for example.

(16) (1) Taking HEK293T cells for example, eukaryotic cells are trained and transfected according to the present invention: HEK293T cells are inoculated and cultured in DMEM sugar culture added by 10% FBS (HyClone, SH30022.01 B), including penicillin (100 U/ml) and streptomycin (100 μg/ml).

(17) (2) Distributing it into a 6-well plate before transfection which is conducted under a density of 70%-80%.

(18) (3) Taking liposome transfection for example. According to manual operation of the Lipofectamine™ 2000 Transfection Reagent (Invitrogen, 11668-019), and taking SpCas9 nickase for example, 2 μg BE3 plasmid and 2 μg pGL3-U6-sgRNA plasmid are evenly blended, and cotransfected into each well. The solution is changed every six to eight hours, and cells are collected after 72 hours.

(19) (4) Analysis of genotype

(20) A, collecting some cells in the pyrolysis liquid (10 μM Tris HCl, 0.4 M NaCl, 2 μM EDTA, 1% SDS) and digesting them by using 100 μg/ml proteinase K. After digestion, phenol-chloroform extraction is made and then dissolved into 50 μl deionized water. B, using a pair of primers N-For and N-Rev for PCR amplification. The PCR recovery product is collected by AxyPrep PCR cleanup purification. 200 ng product is diluted into 20 μl for degeneration and annealing: 95° C., 5 min; 95-85° C. at −2° C./s; 85-25° C. at 0.1° C./s; Hold at 4° C.

(21) C, A base, adenine (A), is added at the end of the PCR recovery product by rTaq. The adenine-added reaction system comprises: 700-800 ng PCR recovery product 5 μl 10×l Buffer (Mg′ PLUS) 4 μl dNTP 0.5 μl rTaq (TAKARA, R001 AM) adding water to obtaion a 50 μl system.

(22) After incubation under 37° C. for 30 minutes, 1 μl product is removed and connected with pMD19-T vector (TAKARA, 3271) to transformate DH5 cells (TransGen, CD201). D, monoclone is selected and the target gene mutation is sequenced with universal primer M13-F. The sequencing results are shown below (the bold and underlined portions represent PAMs; the italics represent mutant codes; and the italic and underlined portions represent mutant bases):

(23) TABLE-US-00002 1. hPD-1 Sg-1: CTACAACTGGGCTGGCGGCCAAG Mut: CTA AACTGGGCTGGCGGCCAAG Sg-2: CAGCAACCAGACGGACAAGCTGG Mut: CAG AACCAGACGGACAAGCTGG Sg-3: CGGCCAGTTCCAAACCCTGGTGG Mut: CGGCTAGTTCCAAACCCTGGTGG 2. hLAG3 Sg-1: CCAGACCATAGGAGAGATGTGGG Mut: CCAGACCATAGGAGAGATGTG G Sg-2: CCATAGGAGAGATGTGGGAGGCT Mut: CCATAGGAGAGATGTG GAGGCT Sg-3: CCGGCGGCGCCCTCCTCCTGGGG Mut: CCAGCGGCGCCCTCCTCCTG GG 3. hTIGIT Sg-1: GATCGAGTGGCCCCAGGTCCCGG Mut: GAT GAGTGGCCCCAGGTCCCGG 4. hVISTA Sg-1: CCTTCTACAAGACGTGGTACCGC Mut: CCTTCTACAAGACGTG TACCGC 5. 2B4 Sg-1: GCAGCTCAGCAGCAGGACAGTGG Mut: GCAGCT AGCAGCAGGACAGTGG 6. hCD160 Sg-1: AAAACAGCTGAGACTTAAAAGGG Mut: AAAA AGCTGAGACTTAAAAGGG

(24) The results show that the target genes result in the target base mutation of sgRNA, the termination codons are introduced, and the gene knockouts of PD1, LAG3, TIGIT, VISTA, 2B4 and CD160 are achieved successfully.

Example 2

(25) In the primary cells, BE3 mediated base editing was conducted, and the termination codon was introduced to realize gene knockout.

(26) The gene knockout of the primitive cells in human T cells is operated regularly (through electrotransfection or liposome transfection), taking electrotransfection for example.

(27) (1) The Separation and Purification of PBMC Cells:

(28) A, using an anticoagulant tube to collect peripheral blood, with the tube being shaken during the collection so as to have the peripheral blood fully mixed with the anticoagulant;

(29) B, mixing peripheral blood cells and lymphocyte separation medium with equal volume, performing centrifugation, and draining white membrane layer of cells obtained after centrifugation;

(30) C, mixing the white membrane layer of cells with PBS or serum-free cell culture medium 1640 and then performing centrifugation. The precipitation is the PBMC cells.

(31) Repeat three times.

(32) (2) Enrichment of the CD3 Positive Cells

(33) A, adjusting PBMC cells concentration to 50×10.sup.6 cell/ml;

(34) B, adding 50 μl CD3+ enriched antibodies cocktail per ml, blending and then standing at room temperature for 5 minutes;

(35) C, adding 150 μl per ml magnet, blending and then standing for 10 minutes at room temperature;

(36) D, placing the centrifugal tube on a magnetic rack and standing for 5 minutes, then draining upper cell suspension into a new 15 ml centrifugal tube.

(37) E, repeat the operation once.

(38) F, performing centrifugation: 300*g at room temperature for 10 minutes and then collecting cells.

(39) G, cell counting.

(40) (3) Electrotransfection of CD3 Positive Cells

(41) A, configure the electrotransfection system

(42) Adding 8 μg BE3 plasmid and 8 μg pGL3-U6-sgRNA plasmid to a 1.5 ml centrifugal tube, then adding 82 μl electrotransfection buffer and 18 μl supplement1 according to the Lonza Amaxa DianZhuan kit specifications, and mixing evenly.

(43) B, collecting 20×10.sup.6 cells to a 15 ml centrifugal tube, centrifuging 300 g for 10 minutes, and discarding the supernatant.

(44) C, resuspending cells with the electrotransfection solution made from A, and transferring it to an eletrotransfection cup.

(45) D, Using the instrument Lonza 2B, U-014 procedures for electrotransfection.

(46) E, after electrotransfection, the cells being removed to a preheated AIM-V medium added with 10% FBS quickly, and incubated in 5% CO′ incubator at 37° C. for 2 hours.

(47) F, changing the solution for cells after electrotransfection, resuspending the cells with 1×10.sup.6/ml cell density, and incubating overnight.

(48) (4) The Activation and Cultivation of T Cells

(49) A, after 24 hours for electrotransfection, adding 100 U/ml IL-2 to the medium, adding CD3/CD28 dynabeads to the proportion of 1:1, and activating T cells.

(50) B, every two days changing half in liquid to the cells, or adding IL-2, so that the cell density is always maintained at 1×106/ml.

(51) C, after being activated for five days the T cells being collected in a 15 ml centrifugal tube which is positioned in a magnetic frame. Slowly removing supernatant to another clean 15 ml centrifugal tube. Repeat this step once.

(52) D, centrifuging 300*g at room temperature for 10 minutes, removing supernatant, and resuspending cells by using 10% FBS, 300 U/ml AIM-IL-2 V medium, with density controlled in 1×106/ml.

(53) E, every two days changing half in liquid to the cells, or adding IL-2, and counting, with cell density always maintained at 1×106/ml.

(54) (5) Analysis of Genotype

(55) A, collecting some cells in the pyrolysis liquid (10 μM Tris HCl, 0.4 M NaCl, 2 μM EDTA, 1% SDS), and digesting them by using 100 μg/ml proteinase K. After digestion, phenol-chloroform extraction is made and then dissolved into 50 μl deionized water.

(56) B, using a pair of primers N-For and N-Rev for PCR amplification. The PCR recovery product is collected by AxyPrep PCR cleanup purification. 200 ng product is diluted to 20 μl for degeneration and annealing: 95° C., 5 min. 95-85° C. at −2° C./s; 85-25° C. at 0.1° C./s; Hold at 4° C.

(57) C, A base, adenine (A), is added at the end of the PCR recovery product by rTaq. The adenine-added reaction system compromises: 700-800 ng PCR recovery product 5 μl 10× Buffer (Mg.sup.2++PLUS) 4 μl dNTP 0.5 μl rTaq (TAKARA, R001 AM) adding water to obtaion a 50 μl system.

(58) After incubation under 37° C. for 30 minutes, 1 μl product is removed and connected with pMD19-T vector (TAKARA, 3271) to transform DH5 competent cells (TransGen, CD201).

(59) D, monoclone is selected, and sequence each T cells target gene mutations with universal primers M13-f. The sequencing results are shown as below (the bold and underlined portions represent PAMs; the italics represent mutant codes; and the italic and underlined portions represent mutant bases):

(60) TABLE-US-00003 1. hPD-1 Sg-1: CTACAACTGGGCTGGCGGCCAGG Mut: CTA AACTGGGCTGGCGGCCAGG Sg-2: CAGCAACCAGACGGACAAGCTGG Mut: CAG AACCAGACGGACAAGCTGG Sg-3: CGGCCAGTTCCAAACCCTGGTGG Mut: CGGCTAGTTCCAAACCCTGGTGG 2. hLAG3 Sg-1: CCAGACCATAGGAGAGATGTGGG Mut: CCAGACCATAGGAGAGATGTG G Sg-2: CCATAGGAGAGATGTGGGAGGCT Mut: CCATAGGAGAGATGTG GAGGCT Sg-3: CCGGCGGCGCCCTCCTCCTGGGG Mut: CCGGCGGCGCCCTCCTCCTG GG 3. hTIGIT Sg-1: GATCGAGTGGCCCCAGGTCCCGG Mut: GAT GAGTGGCCCCAGGTCCCGG 4. hVISTA Sg-1: CCTTCTACAAGACGTGGTACCGC Mut: CCTTCTACAAGACGT GTACCGC 5. 2B4 Sg-1: GCAGCTCAGCAGCAGGACAGTGG Mut: GCAGCT AGCAGCAGGACAGTGG 6. hCD160 Sg-1: AAAACAGCTGAGACTTAAAAGGG Mut: AAAA AGCTGAGACTTAAAAGGG

(61) The results show that the target genes result in the target base mutation of sgRNA, the termination codons are introduced, and the gene knockouts of PD1, LAG3, TIGIT, VISTA, 2B4 and CD160 are achieved successfully.

Example 3

(62) Developing BE3 Mediated Gene Knockout Mice

(63) Conducting regular operation on mice embryo collection, microinjection of embryo, embryo culture and embryo transfer, etc. For example, the mice were knocked out of TIM3 and LAG3 genes. (1) Microinjection: the fertilized egg was injected by BE3 mRNA and TIM3 specificity sgRNA (corresponding to SEQ ID NO:15), or by BE3 mRNA and LAG3 specificity sgRNA (corresponding to SEQ ID NO:16) respectively. Conventional embryo transfer was then conducted; (2) genotype analysis: genomic DNA is extracted by regular mice tail-cutting, and the coding regions are PCR amplified respectively. Sanger sequencing, with the sequencing result shown as below (the bold and underlined portions represent PAMs; the italics represent mutant codes; and the italic and underlined portions represent mutant bases):

(64) TABLE-US-00004 7. mTIM3 Sg-1: CCTCGTGCCCGTCTGCTGGGGCA Mut: CCTCGTGCCCGTCTGCTAGGGCA 8. mLAG3 Sg-1: CCAGACCATAGGAGAGATGTGG Mut: CCAGACCATAGGAGAGATGTGA

(65) The above results demonstrate CT mutation of TIM3 and LAG3 and introduction of termination codon. TIM3 and LAG3 knockout mice have been successfully developed.