COMPOSITION CONTAINING C2CL ENDONUCLEASE FOR DIELECTRIC CALIBRATION AND METHOD FOR DIELECTRIC CALIBRATION USING SAME

Abstract

Provided is a dielectric calibration technique using C2c1 endonuclease. The dielectric calibration technique is characterized by being especially applicable to eukaryotic cells, for example, to mammalian cells.

Claims

1. A genome editing composition, comprising: a C2c1 endonuclease, a gene coding therefor, or an expression vector carrying the gene; and a guide RNA, a DNA coding therefor, or an expression vector carrying the DNA.

2. The genome editing composition of claim 1, wherein the C2c1 endonuclease is derived from Alicyclobacillus acidoterrestris.

3. The genome editing composition of claim 2, wherein the guide RNA is represented by the following General Sequence Formula 1 or is a mutant resulting from the deletion of 1-15 nucleotides from the sequence of General Sequence Formula 1: TABLE-US-00005 5-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCAC UUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUG GX.sub.1X.sub.2X.sub.3[(N)n]-3 (GeneralSequenceFormula1) wherein, X.sub.1, X.sub.2, and X.sub.3 are each a nucleotide independently selected from A, U, G, and C, and (N)n stands for n nucleotides that may be the same or different and are each independently selected from A, U, G, and C, wherein n is an integer of 17-23.

4. The genome editing composition of claim 3, wherein the deletion is performed on 1-15 nucleotides selected from the sequence AGCUUCUCAAA in General Sequence Formula 1.

5. The genome editing composition of claim 3, wherein the guide RNA is represented by the following General Sequence Formula 2: TABLE-US-00006 5-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCAC UUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUG GCAC[(N)n]-3 (GeneralSequenceFormula2) wherein, (N)n represents n nucleotides which may be the same or different and are selected from A, U, G, and C wherein n is an integer of 17-23.

6. The genome editing composition of claim 5, wherein n is 20.

7. The genome editing composition of claim 1, wherein the C2c1 endonuclease further comprises a nuclear localization signal.

8. The genome editing composition of claim 1, wherein the genome editing composition is applied to an eukaryotic cell or an eukaryotic organism.

9. A genome editing method, comprising a step of introducing the genome editing composition of claim 1 into an isolated cell or organisms exclusive of humans.

10. The genome editing method of claim 9, wherein the cell is an eukaryotic cell and the organism is an eukaryotic organism exclusive of humans.

11. An isolated genetically modified cell, having the genome editing composition of claim 1 introduced thereinto.

12. A genetically modified organism, exclusive of a human, having the genome editing composition of claim 1 introduced thereinto.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIGS. 1a to 1c show configurations of AacC2c1-CRISPR system expression vectors and results of the protein expression using the same, presenting schematic diagrams of 1a an AacC2c1 protein expression vector and an Aac-sgRNA cloning vector (1a) and AacC2c1 protein expression results (1b) (left panel: 1.sup.st antibody, HA tag; and right panel: 1.sup.st antibody, GAPDH).

[0061] FIGS. 2a to 2d show human HPRT1 gene editing results obtained by using the AacC2c1 and sgRNA expression vectors, presenting graphs of human HPRT1 target gene editing results (Indel (%)) obtained using AacC2c1 for Hela cells (2a) and HEK293T cells (2b) (Biological replicate (N=3)), 1.sup.st NGS analysis results for HPRT1-TS2 and HPRT1-TS5 (2c) (PAM sequence in blue; AacC2c1 target sequences in upper cases; sequences around the AacC2c1 target in lower cases; and WT, wild type), and photographs of colony formation assay results obtained through crystal violet staining (2d) (after transfection of AacC2c1 and sgRNA expression vectors thereinto, Hela cells were seeded and subjected to 6-thioguanine (6-TG) selection. Two weeks later, colonies of the cells in which a HPRT1 gene was knocked out by mutation were identified by crystal violet staining)

[0062] FIGS. 3a to 3e show gene editing results obtained by applying AacC2c1 and sgRNA expression vectors to human CCR5 and DNMT1 genes, presenting graphs of human CCR5 target gene editing results (Indel (%)) obtained using AacC2c1 for Hela cells (3a) and HEK293T cells (3b) (Biological replicate (N=1)), graphs of human DNMT1 target gene editing results (Indel (%)) obtained using AacC2c1 or Hela cells (3c) and HEK293T cells (3d) (Biological replicate (N=1)), and 1.sup.st NGS analysis results for CCR5-TS3 and DNMT1-TS4 (3e) (PAM sequence in blue; AacC2c1 target sequences in upper cases; sequences around the AacC2c1 target in lower cases; and WT, wild type).

MODE FOR CARRYING OUT THE INVENTION

[0063] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1: Vector Construction

[0064] With pRSFDuet1-His6-Sumo-AacC2C1-CN plasmid (pRSF-Duet-1-His6-SUMO-AacC2c1 plasmid, granted by Pf. Dinshaw Patel group, was added with an NLS(PKKKRKV)-HA tag sequence at the C-terminal of AacC2c1) serving as a template, the coding sequence of AacC2c1 was amplified by PCR (CMV-AacC2c1-CN-1F-Hind3: GCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGCCACCATGGCAGTGAAAAG CATCAAAG (SEQ ID NO: 5), CMV-AacC2c1-CN-1R-BamH1: AACATCGTATGGGTAGGATCCTCAGGCGTAGTCGGGCAC (SEQ ID NO: 6), phusion DNA polymerase was used), and the PCR product was inserted into pcDNA3.1 backbone vector (Invitrogen, V790-20) by Gibson cloning.

[0065] In order to be optimized for human cell expression and genome editing, the above-prepared AacC2c1 coding sequence was used, together with the CMV promoter and the nuclear localization signal (PKKKRKV), to construct an expression cassette. An AacC2c1 expression vector was constructed under the design that the AacC2c1 gene is transcribed under the CMV promoter suitable for human cell expression and the SV40 nucleus localization signal (PKKKRKV) and the HA tag are positioned at the C terminal of the AacC2c1 coding sequence (pcDNA3.1-AacC2c1-NLS-HA; FIG. 1a).

[0066] An Aac-sgRNA cloning vector was constructed in which the sgRNA binding complementarily to a target sequence was transcribed under the human U6 promoter. For Aac-sgRNA, the sequence in the 5-terminal sequence was used in common while 20 consecutive nucleotides at the 3-terminal site of sgRNA were substituted in accordance with target sequences (see Table 2) to construct sgRNA that acted at specific target positions (see General Sequence Formula 2). The Aac-sgRNA cloning vector was constructed on the basis of a U6-Sp-sgRNA cloning vector (see FIG. 1a).

Example 2: Cell Test

[0067] Hela cells (ATCC) and HEK293T cells (ATCC) were both tested in 24-well-plate scales. On the day before transfection, cells were seeded. The next day, transfection was performed at 70-80% confluency. Using lipofectamine2000 (Invitrogen), 500 ng of each of the AacC2c1 and Aac-sgRNA vectors prepared in Example 1 was delivered into the cells. 72 hours after transfection, genomic DNA samples were extracted using DNeasy Blood & Tissue kit (Qiagen) and subjected to the following additional analyses.

Example 3: Targeted Deep Sequencing

[0068] A Deep sequencing library was prepared by PCR. TruSeq HT Dual Index primers were used to assign respective I.D.s to samples, followed by analysis with the aid of MiniSeq 300 cycles kit (Illumina). Primer sequences used in the deep sequencing are summarized in Table 1, below:

TABLE-US-00003 TABLE1 SEQ Primer ID Locus name NO Primersequence Targetsite HPRT1 hHPRT1- 8 gtgctgggattacacgtgtg HPRT1-E2-TS1, E2-1F HPRT1-E2-TS2, hHPRT1- 9 tgattcagccccagtccatt HPRT1-E2-TS3, E2-1R HPRT1-E2-TS4 hHPRT1- 10 ACACTCTTTCCCTACACGACGCTCTT E2- uATCTtgtttgtatcctgtaatgctctca deepF hHPRT1- 11 GTGACTGGAGTTCAGACGTGTGCTC E2- TTCCGATCTcaaatagcaagtactcagaacagc deepR hHPRT1- 12 agttcactccagcctcaaca HPRT1-E2-TS5, E3-1F HPRT1-E2-TS6, hHPRT1- 13 cccaagtcccaacagcaatt HPRT1-E2-TS7, E3-1R HPRT1-E2-TS8 hHPRT1- 14 ACACTCTTTCCCTACACGACGCTCTT E3- CCGATCTggaagtttaatgactaagaggtgtt deepF hHPRT1- 15 GTGACTGGAGTTCAGACGTGTGCTC E3- TTCCGATCTagctcncagtctgataaaatcta deepR ca CCR5 hCCR5- 16 gtttgcattcatggagggca CCR5-TS1, 1-1F CCR5-TS2, hCCR5- 17 gaccagccccaagatgacta CCR5-TS3, 1-1R CCR5-TS4 hCCR5- 18 ACACTCTTTCCCTACACGACGCTCTT 1-deepF CCGATCTcatcctcatcctgataaactgca hCCR5- 19 GTGACTGGAGTTCAGACGTGTGCTC 1-deepR TTCCGATCTcaaacacagcatggacgaca hCCR5- 20 ccttctgggctcactatgct CCR5-TS5, 2-1F CCR5-TS6, hCCR5- 21 gcgtcatcccaagagtctct CCR5-TS7, 2-1R CCR5-TS8 hCCR5- 22 ACACTCTTTCCCTACACGACGCTCTT 2-deepF CCGATCTtttgcgtctctcccaggaat hCCR5- 23 GTGACTGGAGTTCAGACGTGTGCTC 2-deepR TTCCGATCTgatggtgaagataagcctcaca DNMT1 hDNMT 24 ttgggaatgtgggtactgct DNMT1-TS1, 1-1-1F DNMT1-TS2, hDNMT 25 cagaggttacagtgagccga DNMT1-TS3, 1-1-1R DNMT1-TS4 hDNMT 26 ACACTCTTTCCCTACACGACGCTCTT 1-1- CCGATCTTCTtgtgthccttgttctctgacac deepF hDNMT 27 GTGACTGGAGTTCAGACGTGTGCTC 1-1- TTCCGATCTgtttggaaaggggtttgggg deepR hDNMT 28 taggcatgtaccaccacacc DNMT1-TS5, 1-2-1F DNMT1-TS6, hDNMT 29 tcagtgcccaccgataatca DNMT1-TS7, 1-2-1R DNMT1-TS8 hDNMT 30 ACACTCTTTCCCTACACGACGCTCTT 1-2- CCGATCTtgtcaagtgggtgatctct deepF hDNMT 31 GTGACTGGAGTTCAGACGTGTGCTC 1-2- TTCCGATCTtgtagaacttgttgacccgga deepR

Example 4: 6TG Selection

[0069] After transfection of the AacC2c1 vector and the sgRNA vector thereinto in 24-well-plate scales, the cells were passaged to secure a sufficient number. One week after transfection, 1.5X.sub.106 cells were seeded in a 100 pi dish. From the next day, selection was started in a 6 M 6TG (6-thioguanine) condition. Two weeks later, colonies of the cells in which the HPRT1 gene had been knocked out (KO) by mutation were identified by crystal violet staining.

Example 5: Confirmation of AacC2c1 Expression

[0070] Prior to genome editing, examination was made to see whether the AacC2c1 protein was expressed in human cells. In this regard, the pcDNA3.1-AacC2c1-NLS-HA plasmid was transfected into HEK293T cells with the aid of lipofectamine2000 (see Example 4). Twenty four hours after transfection, a whole cell lysate (WCL) was taken and assayed for AacC2c1 expression by a Western blotting method using a 1.sup.st antibody specifically recognizing an HA tag present at the C-terminus of the AacC2c1 protein so as to specifically observe only the AacC2c1 protein.

[0071] The results are depicted in FIG. 1b (left panel: 1.sup.st antibody, HA tag; right panel: 1.sup.st antibody, GAPDH). As shown in FIG. 1b, when the 1.sup.st antibody specifically recognizing an HA tag was used, a protein was detected at 133 kDa in the p3-Aac-C2c1-CN plasmid-transfected experiment group, but not in the mock control.

Example 6: Genome Editing Using AacC2c1

[0072] Because the AacC2c1 protein was identified to be expressed in human cells (HEK293T cells) in Example 5, an examination was made to test whether human genome editing could be conducted with AacC2c1. A human HPRT1 gene was selected as a target gene. Experimental preference was given to HPRT1 gene because when complete KO was introduced by gene editing, a KO clone could be identified on a phenotype basis through 6-thioguanine (6-TG) selection and crystal violet staining.

[0073] AacC2c1 target sequences were selected for human HPRT1 gene and exon sequences of CCR5 and DNMT1 genes (Table 2), and respective Aac-sgRNA vectors corresponding to the target sequences were constructed (see Example 1).

TABLE-US-00004 TABLE2 SEQ TARGETSITE ID (5-nTTN(nPAM)-X.sub.20) NO AacC2c1-HPRT1-TS1 tTTTGCATACCTAATCATTATGCT 32 AacC2c1-HPRT1-TS2 aTTATGCTGAGGATTTGGAAAGGG 33 AacC2c1-HPRT1-TS3 aTTAGGTATGCAAAATAAATCAAG 34 AacC2c1-HPRT1-TS4 tTTCCAAATCCTCAGCATAATGAT 35 AacC2c1-HPRT1-TS5 aTTGTAGCCCTCTGTGTGCTCAAG 36 AacC2c1-HPRT1-TS6 tTTGCTGACCTGCTGGATTACATC 37 AacC2c1-HPRT1-TS7 cTTGAGCACACAGAGGGCTACAAT 38 AacC2c1-HPRT1-TS8 tTTGATGTAATCCAGCAGGTCAGC 39 AacC2c1-CCR5-TS1 tTTCCTTCTTACTGTCCCCTTCTG 40 AacC2c1-CCR5-TS2 cTTACTGTCCCCTTCTGGGCTCAC 41 AacC2c1-CCR5-TS3 aTTTCCAAAGTCCCACTGGGCGGC 42 AacC2c1-CCR5-TS4 gTTGACACATTGTATTTCCAAAGT 43 AacC2c1-CCR5-TS5 tTTCCAGACATTAAAGATAGTCAT 44 AacC2c1-CCR5-TS6 aTTAAAGATAGTCATCTTGGGGCT 45 AacC2c1-CCR5-TS7 cTTGTCATGGTCATCTGCTACTCG 46 AacC2c1-CCR5-TS8 gTTTTTAGGATTCCCGAGTAGCAG 47 AacC2c1-DNMT1-TS1 tTTGTCCTTGGAGAACGGTGCTCA 48 AacC2c1-DNMT1-TS2 gTTCTCCAAGGACAAATCTTTATT 49 AacC2c1-DNMT1-TS3 cTTACAACCGGGAAGTGAATGGAC 50 AacC2c1-DNMT1-TS4 aTTCACTTCCCGGTTGTAAGCATG 51 AacC2c1-DNMT1-TS5 tTTCCGGTAGTGCTCTGGGTACAG 52 AacC2c1-DNMT1-TS6 gTTGCTGCCTTTGATGTAGTCGGA 53 AacC2c1-DNMT1-TS7 aTTGGCCGGATCAAAGAGATCTTC 54 AacC2c1-DNMT1-TS8 cTTCTGTCCCAAGAAGAGCAACGG 55 (X.sub.20 regions corresponding to the sequences except n(PAM) account for target sequences)

[0074] AacC2c1 and Aac-sgRNA expression vectors were delivered into Hela cells and HEK293T cells with the aid of lipofectamine2000. 72 Hours after vector delivery, genomic DNA was isolated, and analyzed by targeted deep sequencing for gene editing efficiency on on-target sequences (introduced indel frequency (%)). For assessing the statistical significance of the introduced indels, targeted deep sequencing was performed on mock controls, which had not been treated with the AacC2c1 and Aac-sgRNA expression vectors. The experiment procedures were repeated three times in total.

[0075] Analysis results are depicted in FIGS. 2a (Hela cell) and 2b (HEK293T cell). As shown in FIGS. 2a and 2b, HPRT1-TS2 and HPRT1-TS5 were respectively observed to introduce indel at frequencies of as high as 6.7% and 5.8% on average into the on-target sequences of Hela cells and at frequencies of as high as 2.8% and 17% on average into the target-sequences of HEK293T cells.

[0076] In addition, targeted deep sequencing raw data were analyzed to examine how indels were mainly introduced into on-target sequences. The results are depicted in FIG. 2c. As can be seen in FIG. 2c, the mutation pattern is deletion that was made mainly at 3 terminal regions of the on-target sequences (FIG. 2c).

[0077] In order to more clearly prove the results obtained by the genetic analysis, the Hela cells to which the AacC2c1 and Aac-sgRNA expression vectors were delivered were subjected to 6-TG selection and crystal violet staining to investigate HPRT1 gene-knockout cells on a phenotype basis. In greater detail, the AacC2c1 and sgRNA expression vectors were transfected into Hela cells which were then seeded and subjected to 6-thioguanine (6-TG) selection. Two weeks later, colonies of the cells in which HPRT1 gene was knocked out by mutation were detected by crystal violet staining. The results are depicted in FIG. 2d. As shown in FIG. 2d, the cells to which the Aac-sgRNA and AacC2c1 plasmids corresponding respectively to HPRT1-TS2 and HPRT1-TS5 were delivered had the HPRT1 gene completely knocked out therein, and thus formed a lot of colonies as detected by 6-TG selection and appeared intensively stained upon crystal violet staining

[0078] Examination was made to investigate whether the AacC2c1-CRISPR-Cas endonucleases could make gene editing on target sequences of genes other than HPRT1 gene. To this end, CCR5 and DNMT1 genes were additionally selected. For each of the target genes, 8 AacC2c1 target sequences were selected (Table 2). Aac-sgRNA vectors corresponding respectively to the target sequence were delivered, together with the AacC2c1 expression plasmid, into Hela cells and HEK293T cells. 72 hours after delivery, genomic DNA was extracted and analyzed for the introduction of indels into on-target sequences to measure indel frequencies.

[0079] The indel frequencies thus obtained are depicted in FIGS. 3a (indel frequencies on CCR5 target regions in Hela cells), 3b (indel frequencies on CCR5 target regions in HEK293T cells), 3c (indel frequencies on DNMT1 target regions in Hela cells), and 3d (indel frequencies on DNMT1 target regions in HEK293T cells). As can be seen in FIGS. 3a to 3d, indels were introduced into the CCR5-TS3 target region at frequencies of 6.9% for Hela and 1.4% for HEK293T cells and into the DNMT1-TS4 region at frequencies of 2.1% for Hela cells and 3.1% for HEK293T cells.

[0080] In addition, 1.sup.st NGS (targeted deep sequencing) was performed for CCR5-TS3 and DNMT1-TS4 and the result is depicted in FIG. 3e. As shown in FIG. 3e, both CCR5-TS3 and DNMT1-TS4 were observed to have indels introduced into the target sequences therein.

[0081] Based on phenotype analysis results, genetic analysis confirmed that the wild-type AacC2c1-CRISPR system can be used for human genome editing and operates for specific target sequence locations at high yield.