METHOD AND COMPOSITION FOR A TARGETED GENE KNOCKOUT

Abstract

A composition is for the targeted knockout of a gene on double-stranded DNA in a biological cell. A method is for the targeted knockout of a gene on double-stranded DNA in a biological cell. A preparation includes a biological cell prepared in vitro. The biological cell includes a gene on double-stranded DNA, which is knocked-out in a targeted manner. A kit is for the targeted knockout of a gene on double-stranded DNA in a biological cell. Another method is for treating a subject afflicted with a disease associated with a mutated gene. Nucleic acid molecules can be a component of the composition and methods.

Claims

1. A composition for the targeted knockout of a gene on double-stranded DNA in a biological cell, comprising: a first CRISPR endonuclease or a nucleic acid molecule encoding the said CRISPR endonuclease; a second CRISPR endonuclease or a nucleic acid molecule encoding said second CRISPR endonuclease; said first and said second CRISPR endonucleases are configured to create single-strand DNA breaks, a first single guide RNA (sgRNA), and a second sgRNA, said first sgRNA is configured to hybridize to the sense strand of a genetic element controlling the expression of said gene, and said second sgRNA is configured to hybridize to the antisense strand of said genetic element controlling the expression of said gene.

2. The composition of claim 2, wherein said genetic element controlling the expression of said gene is a promoter.

3. The composition of claim 1, wherein said first or second CRISPR endonuclease is a variant of CRISPR associated protein 9 (Cas9).

4. The composition of claim 3, wherein said variant of Cas9 comprises mutation(s) in the nuclease domain(s) RuvC or HNH, said mutation(s) conferring DNA nickase activity.

5. The composition of claim 3, wherein said variant of Cas 9 is Cas9 D10A nickase or Cas9 H840A nickase.

6. The composition of claim 1, wherein said gene is a mutated form of a wild type gene (mutated gene).

7. The composition of claim 6, wherein said mutated gene is selected from the group consisting of: gain-of-function mutated gene, disease-associated mutated gene, mutated non-essential gene, and a mutated form of the gene encoding neutrophil elastase (ELANE).

8. The composition of claim 1, wherein said first or second sgRNA comprises the nucleotide sequence which is selected from the group consisting of SEC ID NOS: 1 to 252.

9. A method for the targeted knockout of a gene on double-stranded DNA in a biological cell, comprising the following steps: 1) providing a biological cell comprising a gene on double-stranded DNA; 2) introducing into said biological cell a composition comprising: a first CRISPR endonuclease or a nucleic acid molecule encoding the said CRISPR endonuclease; a second CRISPR endonuclease or a nucleic acid molecule encoding said second CRISPR endonuclease; said first and said second CRISPR endonucleases are configured to create single-strand DNA breaks, a first single guide RNA (sgRNA), and a second sgRNA, said first sgRNA is configured to hybridize to the sense strand of a genetic element controlling the expression of said gene, and said second sgRNA is configured to hybridize to the antisense strand of said genetic element controlling the expression of said gene, and 3) incubating said cell and said composition, thereby allowing the creation of a single-strand DNA break on the sense strand of a genetic element controlling the expression of said gene and a single-strand DNA break on the antisense strand of the genetic element controlling the expression of said gene.

10. The method of claim 9, wherein said composition is the composition of claim 1.

11. The method of claim 9, wherein said biological cell is a primary cell or a hematopoietic stem and progenitor cell (HSPC).

12. The method of claim 11, wherein said biological cell originates from a subject with a mutated gene.

13. The method of claim 12, wherein said mutated gene is selected from the group consisting of: gain-of-function mutated gene, disease-associated mutated gene, mutated non-essential gene, and mutated form of the gene encoding neutrophil elastase (ELANE).

14. A preparation comprising a biological cell prepared in vitro by a method comprising the following steps: 1) providing a biological cell comprising a gene on double-stranded DNA; 2) introducing into said biological cell a composition comprising: a first CRISPR endonuclease or a nucleic acid molecule encoding the said CRISPR endonuclease; a second CRISPR endonuclease or a nucleic acid molecule encoding said second CRISPR endonuclease; said first and said second CRISPR endonucleases are configured to create single-strand DNA breaks, a first single guide RNA (sgRNA), and a second sgRNA, said first sgRNA is configured to hybridize to the sense strand of a genetic element controlling the expression of said gene, and said second sgRNA is configured to hybridize to the antisense strand of said genetic element controlling the expression of said gene; 3) incubating said cell and said composition, thereby allowing the creation of a single-strand DNA break on the sense strand of a genetic element controlling the expression of said gene and a single-strand DNA break on the antisense strand of the genetic element controlling the expression of said gene, and 4) recovering said cell.

15. A kit for the targeted knockout of a gene on double-stranded DNA in a biological cell, comprising the composition of claim 1 and instructions for delivering the composition to said biological cell so as to knockout the gene.

16. A method of treating a subject afflicted with a disease associated with a mutated gene, comprising administration of a therapeutically effective amount of the composition of claim 1.

17. A nucleic acid molecule comprising a nucleotide sequence which is selected from the group consisting of SEQ ID NOS: 1 to 252.

18. The nucleic acid molecule of claim 17, which is an RNA molecule.

19. A method of treating a subject afflicted with a disease associated with a mutated gene, comprising inhibiting in said subject a genetic element controlling the expression of said gene.

20. The method of claim 19, wherein said inhibiting is carried out by knocking-out said genetic element controlling the expression of said gene.

21. The method of claim 19, wherein said subject is afflicted with a dominant-autosomal disease or an autosomal-recessive disorder.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0107] FIG. 1: Safe and efficient strategy to disrupt the ELANE gene expression by promoter knockout using CRISPR/Cas9 D10A nickase restores granulocytic differentiation of congenital neutropenia patient's HSPCs with p.A57V CN mutation. A, Scheme of the experiment. B, FACS gating strategy for cells generating during in vitro granulocytic differentiation C, Representative FACS density plots for granulocytic CD45.sup.+CD11b.sup.+CD15.sup.+, CD45.sup.+CD15.sup.+CD16.sup.+ and CD45.sup.+CD16b.sup.+C066b.sup.+ cell subsets of differentiated primary HSPCs of congenital neutropenia patient with p.A57V mutation after CRISPR/Cas9 D10A double nickases editing.

[0108] FIG. 2: ELANE promoter knockout restored granulocytic differentiation of congenital neutropenia patient's HSPCs with p.A57V ON mutation. A-D, Granulocytic differentiation capacity of ELANE promoter knockout HSPCs of congenital neutropenia patient's with p.A57V mutation was assessed by liquid culture differentiation after 13 days by investigating neutrophilic surface marker expression. Percentage (A), absolute cell counts (B), morphologic analysis of cytospin slides (C) and representative images of Wright-Giemsa stained cytopsins (D) are shown, E. Colony forming unit assay of the ELANE promoter knockout cells showed higher numbers of CFU-G compared to control cells. Data represent meansstandard deviation (SD) from triplicates. *P<0.05, **P<0.01, ****P<0.0001.

[0109] FIG. 3: ELANE promoter knockout significantly impaired the ELANE gene expression and improved the functionality of differentiated neutrophils. A, ROS production was significantly elevated in promoter knockout cells treated with fMLP, as compared to non-target Cas9 cells. B, ELANE mRNA expression in in vitro generated CN neutrophils was 5-fold reduced compared to unedited healthy donor neutrophils. C, Indel's prevalence was assessed by inference of CRISPR edits (ICE) at day 13 post differentiation and was 87%.

[0110] FIG. 4: Independent in silico on- and off-target profiling of sgRNA confirms the safety of the selected sgRNAs. Independent in silica on- and off-target profiling of each sgRNA was done using Doench 2016 algorithm using CRISPRitz tool for up to 4 mismatches using reference genome GRCh37 (hg19) for sgRNA 2 (A) and sgRNA 30 (B) shows low off-target profile for each sgRNA.

[0111] FIG. 5: Analyzing ELANE promoter knockout genome editing outcome by Next generation sequencing. A, Quantification of editing frequency determined by the percentage and number of sequence reads showing unmodified and modified alleles. B, Nucleotide distribution across amplicon. At each base in the reference amplicon, the percentage of each base as observed in sequencing reads is shown (A=green; C=orange; G=yellow; T=purple). Black bars show the percentage of reads for which that base was deleted. Brown bars between bases show the percentage of reads having an insertion at that position. Sequence is listed as SEQ ID NO: 259. C, Combined frequency of any modification across the amplicon. Modifications outside of the quantification window are also shown.

[0112] FIG. 6: ELANE promoter knockout did NOT impair granulocytic differentiation of healthy control's HSPCs. Granulocytic differentiation capacity of ELANE promoter knockout HSPCs of healthy controls was assessed by liquid culture differentiation after 14 days by investigating neutrophilic surface marker expression and ELANE. PRTN3 and AZU1 mRNA expression in in vitro generated neutrophils from (A), healthy control 98 and (B), healthy control 99. Data represent meansstandard deviation (SD) from triplicates. *P<0.05, **P<0.01, ****P<0.0001.

[0113] FIG. 7: Unbiased genome-wide profiling of off-target cleavage by CRISPR-Cas9 double nickase ELANE promoter knockout using GUIDE-seq. Profiling of off-target cleavage upon double nickase ELANE promoter knockout treatment using GUIDEseq showed only 2 potential off-target sites, LINC00992Long Intergenic Non-Protein Coding RNA 992- and chr9:93703739a non-coding intergenic region-. The ELANE promoter target sequence is shown in the top line with cleaved sites shown underneath and with mismatches to the on-target site shown and highlighted in color. GUIDE-seq sequencing read counts are shown to the right of each site. The on-target site is marked with a green circle and off-target sites with red circle in (A), sgRNA 30 (upper sequence Reads is listed as SEQ ID NO: 260) and (B), sgRNA 2 (upper sequence Reads is listed as SEQ ID NO: 261). All the genomic coordinates are based on reference genome GRCh37 (hg19).

EXAMPLES

1. Material and Methods

Patients

[0114] One severe congenital neutropenia patient harboring an ELANE mutation (ELANE-CN) was used in the study. Healthy individuals served as control group. Bone marrow and peripheral blood samples were collected from the patient in association with an annual follow-up recommended by the Severe Chronic Neutropenia International Registry. Study approval was obtained from the Ethical Review Board of the Medical Faculty, University of Tbingen. Informed written consent was obtained from the participant of this study.

Cell Culture

[0115] Human CD34.sup.+ HSPC were isolated from bone marrow mononuclear cells by magnetic bead separation using human CD34 progenitor cell isolation kit. (Miltenyi Biotech, #130-046-703). CD34.sup.+ cells were cultured in a density of 210.sup.5 cells/ml in Stemline II hematopoietic stem cell expansion medium (Sigma Aldrich, #50192) supplemented with 10% FBS. 1% penicillin/streptomycin, 1% L-glutamine and a cytokine cocktail consisting of 20 ng/ml IL-3, 20 ng/ml IL-6. 20 ng/ml TPO, 50 ng/ml SCF and 50 ng/mL FLT-3L (all cytokines were purchased from R&D Systems).

Design of the sgRNAs

[0116] Specific D10A nickase-guide-RNAs (sgRNAs) to target promoter region of ELANE gene (nick sites: chr19 [GGGCTATAAGAGGAGCCGGG (SEQ ID NO: 1; sgRNA 2), GAGGCCGTTGCATTGCCCCA (SEQ ID NO: 2; sgRNA 30)] were designed by using the Desktop genetics website. In total 252 sgRNAs specific for the ELANE promoter were developed by the inventors. They are listed in the following table along with their genomic positions according to the Genome Reference Consortium Human Build 37 (GRCh37), the specificity for the sense (1) or antisense strand (1) and the protospacer adjacent motif (PAM).

TABLE-US-00001 TABLE1 sgRNAsspecificforthehuman ELANEpromoter Genomic SEQID position NO. (hg19) Strand Sequence PAM 1 852285 1 GGGCTATAAGAGGAGCCGGG CGG 2 852243 1 GAGGCCGTTGCATTGCCCCA CGG 3 851023 1 CTCACTTTCACCCAGGCTCA CGG 4 851030 1 CGGGGAACTCACTTTCACCC AGG 5 851045 1 GGTGAAAGTGAGTTCCCCGT TGG 6 851048 1 GAAAGTGAGTTCCCCGTTGG AGG 7 851048 1 CTCGTCTGTTGCCTCCAACG GGG 8 851049 1 CCTCGTCTGTTGCCTCCAAC GGG 9 851050 1 TCCTCGTCTGTTGCCTCCAA CGG 10 851060 1 CCCGTTGGAGGCAACAGACG AGG 11 851065 1 TGGAGGCAACAGACGAGGAG AGG 12 851069 1 GGCAACAGACGAGGAGAGGA TGG 13 851073 1 ACAGACGAGGAGAGGATGGA AGG 14 851078 1 CGAGGAGAGGATGGAAGGCC TGG 15 851085 1 AGGGCTCATTCTTGGGGGCC AGG 16 851090 1 ACCTCAGGGCTCATTCTTGG GGG 17 851091 1 AACCTCAGGGCTCATTCTTG GGG 18 851092 1 GAACCTCAGGGCTCATTCTT GGG 19 851093 1 TGAACCTCAGGGCTCATTCT TGG 20 851100 1 GCCCCCAAGAATGAGCCCTG AGG 21 851104 1 CAGCCGCTCCCTGAACCTCA GGG 22 851105 1 CCAGCCGCTCCCTGAACCTC AGG 23 851106 1 AAGAATGAGCCCTGAGGTTC AGG 24 851107 1 AGAATGAGCCCTGAGGTTCA GGG 25 851112 1 GAGCCCTGAGGTTCAGGGAG CGG 26 851116 1 CCTGAGGTTCAGGGAGCGGC TGG 27 851126 1 AGGGAGCGGCTGGAGTGAGC CGG 28 851134 1 GCTGGACGGAGATCTGGGGC CGG 29 851138 1 CGCAGCTGGACGGAGATCTG GGG 30 851139 1 CCGCAGCTGGACGGAGATCT GGG 31 851140 1 CCCGCAGCTGGACGGAGATC TGG 32 851148 1 CTCTGGGACCCGCAGCTGGA CGG 33 851150 1 CCCAGATCTCCGTCCAGCTG CGG 34 851151 1 CCAGATCTCCGTCCAGCTGC GGG 35 851152 1 AGGCCTCTGGGACCCGCAGC TGG 36 851160 1 CGTCCAGCTGGGGGTOCCAG AGG 37 851164 1 CGAGTGTAACCCAGGCCTCT GGG 38 851165 1 AGCTGCGGGTCCCAGAGGCC TGG 39 851165 1 GCGAGTGTAACCCAGGCCTC TGG 40 851166 1 GCTGCGGGTCCCAGAGGCCT GGG 41 851172 1 AGGAGCTGCGAGTGTAACCC AGG 42 851185 1 TGGGTTACACTCGCAGCTCC TGG 43 851186 1 GGGTTACACTCGCAGCTCCT GGG 44 851187 1 GGTTACACTCGCAGCTCCTG GGG 45 851188 1 GTTACACTCGCAGCTCCTGG GGG 46 851191 1 ACACTCGCAGCTCCTGGGGG AGG 47 851192 1 GCACGTCAAGGGCCTCCCCC AGG 48 851203 1 TGGGAACTGAGGCACGTCAA GGG 49 851204 1 TTGGGAACTGAGGCACGTCA AGG 50 851214 1 GGGTTCCTGTTTGGGAACTG AGG 51 851220 1 ACGTGCCTCAGTTOCCAAAC AGG 52 851222 1 CCTTCCCAGGGTTCCTGTTT GGG 53 851223 1 TCCTTCCCAGGGTTCCTGTT TGG 54 851228 1 CAGTTCCCAAACAGGAACCC TGG 55 851229 1 AGTTCCCAAACAGGAACCCT GGG 56 851233 1 CCCAAACAGGAACCCTGGGA AGG 57 851234 1 CACTTCTCTGGTCCTTOCCA GGG 58 851235 1 GCACTTCTCTGGTCCTTCCC AGG 59 851246 1 CTGCGCAATAGGCACTTCTO TGG 60 851257 1 TCGGGCACTCACTGCGCAAT AGG 61 851275 1 CGGCCACATGCAGCTGTGTC GGG 62 851276 1 CCGGCCACATGCAGCTGTGT CGG 63 851283 1 GTGCCCGACACAGCTGCATG TGG 64 851287 1 CCGACACAGCTGCATGTGGC CGG 65 851295 1 TACCCAGGGCCCTGTGATAC CGG 66 851296 1 CTGCATGTGGCCGGTATCAC AGG 67 851297 1 TGCATGTGGCCGGTATCACA GGG 68 851303 1 TGGCCGGTATCACAGGGCCC TGG 69 851304 1 GGCCGGTATCACAGGGCCCT GGG 70 851309 1 CGCCTGCCTCAGTTTACCCA GGG 71 851310 1 TCGCCTGCCTCAGTTTACCO AGG 72 851314 1 ACAGGGCCCTGGGTAAACTG AGG 73 851318 1 GGCCCTGGGTAAACTGAGGC AGG 74 851336 1 GCAGGCGACACAGCTGCATG TGG 75 851340 1 GCGACACAGCTGCATGTGGC CGG 76 851348 1 TACCCAGGGCCCTGTGATAC CGG 77 851349 1 CTGCATGTGGCCGGTATCAC AGG 78 851350 1 TGCATGTGGCCGGTATCACA GGG 79 851356 1 TGGCCGGTATCACAGGGCCC TGG 80 851357 1 GGCCGGTATCACAGGGCCCT GGG 81 851362 1 CGCCTGCCTCAGTTTACCCA GGG 82 851363 1 TCGCCTGCCTCAGTTTACCC AGG 83 851367 1 ACAGGGCCCTGGGTAAACTG AGG 84 851371 1 GGCCCTGGGTAAACTGAGGC AGG 85 851389 1 GCAGGCGACACAGCTGCATG TGG 86 851393 1 GCGACACAGCTGCATGTGGC CGG 87 851401 1 TACCCAGGGCCCTGTGATAC CGG 88 851402 1 CTGCATGTGGCCGGTATCAC AGG 89 851403 1 TGCATGTGGCCGGTATCACA GGG 90 851409 1 TGGCCGGTATCACAGGGCCC TGG 91 851410 1 GGCCGGTATCACAGGGCCCT GGG 92 851415 1 CGCCTGCCTCAGTTTACCCA GGG 93 851416 1 TCGCCTGCCTCAGTTTACCC AGG 94 851420 1 ACAGGGCCCTGGGTAAACTG AGG 95 851424 1 GGCCCTGGGTAAACTGAGGC AGG 96 851442 1 GCAGGCGACACAGCTGCATG TGG 97 851454 1 GCTGCATGTGGCCGTATCAC AGG 98 851454 1 TTACCCAGGGCCCTGTGATA CGG 99 851455 1 CTGCATGTGGCCGTATCACA GGG 100 851461 1 GTGGCCGTATCACAGGGCCC TGG 101 851462 1 TGGCCGTATCACAGGGCCCT GGG 102 851467 1 CACCTGCCTCAGTTTACCCA GGG 103 851468 1 TCACCTGCCTCAGTTTACCC AGG 104 851472 1 ACAGGGCCCTGGGTAAACTG AGG 105 851476 1 GGCCCTGGGTAAACTGAGGC AGG 106 851494 1 GCAGGTGACACAGCTGCATG TGG 107 851498 1 GTGACACAGCTGCATGTGGC CGG 108 851506 1 GCTGCATGTGGCCGGTATCA CGG 109 851506 1 TATCCAGGGCCCCGTGATAC CGG 110 851507 1 CTGCATGTGGCCGGTATCAC GGG 111 851508 1 TGCATGTGGCCGGTATCACG GGG 112 851514 1 TGGCCGGTATCACGGGGCCC TGG 113 851520 1 CGCCTGCCTCTGTTTATCCA GGG 114 851521 1 TCGCCTGCCTCTGTTTATCC AGG 115 851525 1 ACGGGGCCCTGGATAAACAG AGG 116 851529 1 GGCCCTGGATAAACAGAGGC AGG 117 851547 1 GCAGGCGACACAGCTGCATG TGG 118 851551 1 GCGACACAGCTGCATGTGGC CGG 119 851559 1 GCTGCATGTGGCCGGTATCA CGG 120 851559 1 TACCCAGGGCCCCGTGATAC CGG 121 851560 1 CTGCATGTGGCCGGTATCAC GGG 122 851561 1 TGCATGTGGCCGGTATCACG GGG 123 851567 1 TGGCCGGTATCACGGGGCCC TGG 124 851568 1 GGCCGGTATCACGGGGCCCT GGG 125 851573 1 CGCCTGCCTCAGTTTACCCA GGG 126 851574 1 TCGCCTGCCTCAGTTTACCC AGG 127 851578 1 ACGGGGCCCTGGGTAAACTG AGG 128 851582 1 GGCCCTGGGTAAACTGAGGC AGG 129 851587 1 TGGGTAAACTGAGGCAGGCG AGG 130 851599 1 CCTGAGGGACTTGATGGGGG TGG 131 851602 1 AGACCTGAGGGACTTGATGG GGG 132 851603 1 TAGACCTGAGGGACTTGATG GGG 133 851604 1 CTAGACCTGAGGGACTTGAT GGG 134 851605 1 CCTAGACCTGAGGGACTTGA TGG 135 851610 1 CCACCCCCATCAAGTCCCTC AGG 136 851614 1 CCTGCCAAACCTAGACCTGA GGG 137 851615 1 ACCTGCCAAACCTAGACCTG AGG 138 851616 1 CCATCAAGTCCCTCAGGTCT AGG 139 851621 1 AAGTCCCTCAGGTCTAGGTT TGG 140 851625 1 CCCTCAGGTCTAGGTTTGGC AGG 141 851630 1 AGGTCTAGGTTTGGCAGGTT TGG 142 851651 1 GGCAAAAACACAGCAACGCT CGG 143 851668 1 GCTCGGTTAAATCTGAATTT CGG 144 851669 1 CTCGGTTAAATCTGAATTTC GGG 145 851683 1 AATTTCGGGTAAGTATATCC TGG 146 851684 1 ATTTCGGGTAAGTATATCCT GGG 147 851690 1 GTCTCTTCCAAATGAGGCCC AGG 148 851694 1 AGTATATCCTGGGCCTCATT TGG 149 851696 1 ATCTAAGTCTCTTCCAAATG AGG 150 851735 1 AAAAAACGTCGAGACCAGCC CGG 151 851738 1 TTTCACCGTGTTGGCCGGGC TGG 152 851742 1 GGGGTTTCACCGTGTTGGCC GGG 153 851743 1 CGGGGTTTCACCGTGTTGGC CGG 154 851744 1 CGAGACCAGCCCGGCCAACA CGG 155 851747 1 GAGACGGGGTTTCACCGTGT TGG 156 851761 1 TTGTATTTTTAGTAGAGACG GGG 157 851762 1 TTTGTATTTTTAGTAGAGAC GGG 158 851763 1 TTTTGTATTTTTAGTAGAGA CGG 155 851785 1 AAAAATACAAAAAATTAGCC AGG 160 851792 1 CAGGCGTGAGCCACTGCGCC TGG 161 851793 1 AAAAAATTAGCCAGGOGCAG TGG 162 851811 1 TCCCAGAGTGCTGGGATCAC AGG 163 851819 1 CCTCAGCCTCCCAGAGTGCT GGG 164 851820 1 CGCCTGTGATCCCAGCACTC TGG 165 851820 1 GCCTCAGCCTOCCAGAGTGC TGG 166 851821 1 GCCTGTGATCCCAGCACTCT GGG 167 851824 1 TGTGATCCCAGCACTCTGGG AGG 168 851830 1 CCCAGCACTCTGGGAGGCTG AGG 169 851834 1 GCACTCTGGGAGGCTGAGGC AGG 170 851837 1 CTCTGGGAGGCTGAGGCAGG CGG 171 851848 1 TGAGGCAGGCGGATCACCCG AGG 172 851853 1 GGTCTTGAACATCTGACCTC GGG 173 851854 1 TGGTCTTGAACATCTGACCT CGG 174 851871 1 TCAGATGTTCAAGACCAGCC TGG 175 851874 1 TTTCGCCCTGTCGGCCAGGC TGG 176 851878 1 AGTGTTTCGCCCTGTCGGCC AGG 177 851879 1 TCAAGACCAGCCTGGCCGAC AGG 178 851880 1 CAAGACCAGCCTGGCCGACA GGG 179 851883 1 GAGACAGTGTTTCGCCCTGT CGG 180 851919 1 CTACAAATACAAAAATTAGC CGG 181 851920 1 TACAAATACAAAAATTAGCC GGG 182 851925 1 ATACAAAAATTAGCCGGGAG TGG 183 851927 1 CAGGCACCTGOCACCACTCC CGG 184 851928 1 CAAAAATTAGCCGGGAGTGG TGG 185 851932 1 AATTAGCCGGGAGTGGTGGC AGG 186 851946 1 TCCTGAATAGCTGAGATTAC AGG 187 851956 1 GCCTGTAATCTCAGCTATTC AGG 188 851959 1 TGTAATCTCAGCTATTCAGG AGG 189 851965 1 CTCAGCTATTCAGGAGGCTG AGG 190 851969 1 GCTATTCAGGAGGCTGAGGC AGG 191 851987 1 GCAGGAGAATCACTTGAACC TGG 192 851988 1 CAGGAGAATCACTTGAACCT GGG 193 851991 1 GAGAATCACTTGAACCTGGG AGG 194 851994 1 AATCACTTGAACCTGGGAGG CGG 195 851994 1 CACGGCAACCTCCGCCTCCC AGG 196 851997 1 CACTTGAACCTGGGAGGCGG AGG 197 852011 1 AGGCGGAGGTTGCCGTGAGC CGG 198 852012 1 GGCGGAGGTTGCCGTGAGCC GGG 199 852012 1 GGTGGCGTGATCCCGGCTCA CGG 200 852019 1 GGAGTGCGGTGGCGTGATCC CGG 201 852030 1 TCGCCCAGGCTGGAGTGCGG TGG 202 852033 1 CTATCGCCCAGGCTGGAGTG CGG 203 852037 1 CACGCCACCGCACTCCAGCC TGG 204 852038 1 ACGCCACCGCACTCCAGCCT GGG 205 852040 1 TCTTGCTCTATCGCCCAGGC TGG 206 852044 1 AGAGTCTTGCTCTATCGCCC AGG 207 852071 1 GGTTTTTTATTTATTTTTT TGG 208 852092 1 AAATGTCAGATAATCAATGT GGG 209 852093 1 CAAATGTCAGATAATCAATG TGG 210 852131 1 GGGGCCTCCAGACAAAATTC AGG 211 852135 1 TGTGCATCCTGAATTTTGTC TGG 212 852138 1 GCATCCTGAATTTTGTCTGG AGG 213 852150 1 ACGCTGGATTGGCTCGGGTG GGG 214 852151 1 GACGCTGGATTGGCTCGGGT GGG 215 852152 1 AGACGCTGGATTGGCTCGGG TGG 216 852155 1 ACAAGACGCTGGATTGGCTC GGG 217 852156 1 GACAAGACGCTGGATTGGCT CGG 218 852161 1 AGGGGGACAAGACGCTGGAT TGG 219 852166 1 GGAGAAGGGGGACAAGACGC TGG 220 852178 1 TGATGAAAAGGGGGAGAAGG GGG 221 852179 1 TTGATGAAAAGGGGGAGAAG GGG 222 852180 1 GTTGATGAAAAGGGGGAGAA GGG 223 852181 1 CGTTGATGAAAAGGGGGAGA AGG 224 852187 1 ACAGGGCGTTGATGAAAAGG GGG 225 852188 1 CACAGGGCGTTGATGAAAAG GGG 226 852189 1 GCACAGGGCGTTGATGAAAA GGG 227 852190 1 GGCACAGGGCGTTGATGAAA AGG 228 852204 1 TTTCATCAACGCCCTGTGCC AGG 229 852204 1 ACTTCCTCTCCCCTGGCACA GGG 230 852205 1 TTCATCAACGCCCTGTGCCA GGG 231 852205 1 CACTTCCTCTCCCCTGGCAC AGG 232 852206 1 TCATCAACGCCCTGTGCCAG GGG 233 852211 1 AACGCCCTGTGCCAGGGGAG AGG 234 852211 1 GCCCTCCACTTCCTCTCCCC TGG 235 852217 1 CTGTGCCAGGGGAGAGGAAG TGG 236 852220 1 TGCCAGGGGAGAGGAAGTGG AGG 237 852221 1 GCCAGGGGAGAGGAAGTGGA GGG 238 852227 1 GGAGAGGAAGTGGAGGGCGC TGG 239 852231 1 AGGAAGTGGAGGGCGCTGGC CGG 240 852237 1 TGGAGGGCGCTGGCCGGCCG TGG 241 852238 1 GGAGGGCGCTGGCCGGCCGT GGG 242 852239 1 GAGGGCGCTGGCCGGCCGTG GGG 243 852239 1 CCGTTGCATTGCCCCACGGC CGG 244 852250 1 CCGGCCGTGGGGCAATGCAA CGG 245 852262 1 TCTTATAGCCCTGTGCTGGG AGG 246 852264 1 ATGCAACGGCCTCCCAGCAC AGG 247 852265 1 TGCAACGGCCTCCCAGCACA GGG 248 852265 1 TCCTCTTATAGCCCTGTGCT GGG 249 852266 1 CTCCTCTTATAGCCCTGTGC TGG 250 852275 1 TCCCAGCACAGGGCTATAAG AGG 251 852281 1 CACAGGGCTATAAGAGGAGC CGG 252 852282 1 ACAGGGCTATAAGAGGAGCC GGG

CRISPR/Cas9 D10A Nickase-gRNA RNP Mediated ELANE Promoter KO in HSPC

[0117] Nucleofection was carried out using the Amaxa nucleofection system (P3 primary kit, #V4XP-3024) according to the manufacturer's instructions. For 110.sup.6 human CD34.sup.+ HSPC, 300 pmol sgRNA and 300 pmol Hifi Cas9 protein (Integrated DNA Technologies) was used for nucleofection.

Assessing Genome Editing Efficiency

[0118] Gene editing efficiency assessed either by Sanger sequencing of the promoter region qRT-PCR.

Assessing Gene Editing Efficiency by NGS

[0119] rhAmpSeq CRISPR analysis system approach was used to accurately estimate the on-target editing efficiency. The IDT's rhAmpSeq design tool was used to design rhAMP PCR primers to amplify the target area in ELANE promoter. The PCR amplification followed by rhAmpSeq library preparation was performed using rhAmpSeq CRISPR Library Kit (IDT, #10007317) according to manufacturer's protocol. The rhAmpSeq libraries was sequenced on an Illumina platform by Novogene. The NGS data was analysed by IDT's rhAmpSeq CRISPR analysis pipeline followed by CRISPRESSO tool.

RNA Isolation, cDNA Synthesis and qRT-PCR

[0120] RNA was isolated using RNeasy Mini Kit (Qiagen) and cDNA was prepared using Omniscript RT kit (Qiagen). qPCR was performed using SYBR Green qPCR master mix (Roche) and Light Cycler 480 (Roche). ELANE [primers: Fwd: GTGTCWTCCTCGCCTGTGTC (SEQ ID NO: 253), Rev: CCCACAATCTCCGAGGCCAG (SEQ ID NO: 254)] mRNA expression was normalized to GAPDH [primers: Fwd: CTGGGCTACACTGAGCACC (SEQ ID NO: 255), Rev: AAGTGGTCGTTGAGGGCAATG (SEQ ID NO: 256)] and for ACTB [primers: Fwd: CATGTACGTTGCTATCCAGGC (SEQ ID NO: 257), Rev: CTCCTTAATGTCACGCACGAT (SEQ ID NO: 258)] mRNA expression levels.

GUIDEseq Off-Target Profiling

[0121] GUIDEseq was used to detect potential off-target cleavage sites after CRISPR/Cas9 editing. Primary human bone-marrow derived CD34+ cells were cultured in a density of 2105 cells/mL in Stemline II hematopoietic stem cell expansion medium (Sigma Aldrich, #50192) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine and a cytokine cocktail consisting of 20 ng/mL IL-3, 20 ng/mL IL-6, 20 ng/mL TPO, 50 ng/ml SCF and 50 ng/mL FLT-3L (all cytokines were purchased from R&D Systems). CD34+ cells were electroporated with 8 g Cas9 D10A Nickase V3 (IDT, #1081063) assembled with 195 ng sgRNA 2 (IDT) and 195 ng sgRNA 30 (IDT). dsODN was added at a final concentration of 1.5 M (5-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3 (SEQ ID 262), 5-P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C -3 (SEQ ID 263) using the electroporation program CA-137 in 20 l P3 buffer (Lonza, #V4XP-3032) on a Lonza 4-D nucleofector device according to the manufacturer's instruction. 96 hours post-electroporation, the genomic DNA was isolated using QIAamp DNA Blood Mini Kit (Qiagen, #51104) according to manufacturers recommendations. Isolated DNA was sheared with Covaris S200 instrument to an average length of DNA pieces of 500 bp. Sheared DNA was end-repaired, A-tailed and ligated to half-functional adapters, incorporating a 8-nt random molecular index. Two rounds of nested anchored PCR with primers complementary to the oligo tag were used for target enrichment. The final library was sequenced on an Illumina platform by Novogene. The NGS results were analyzed by GUIDEseq analysis pipeline.

Liquid Culture differentiation of CD34+ Cells

[0122] CD34+ cells were seeded in a density of 210.sup.5 cells/ml. Cells were incubated for 7 days in RPMI 1640 GlutaMAX supplemented with 10% FBS. 1% penicillin/streptomycin, 5 ng/ml SCF, 5 ng/ml IL-3, 5 ng/ml GM-CSF and 10 ng/ml G-CSF. Medium was exchanged every second day. On day 7, cells were plated in RPMI 1640 GlutaMAX supplemented with 10% FBS, 1% penicillin/streptomycin and 10 ng/ml G-CSF. On day 14, cells were analyzed by FACS using following mouse anti-human anti-bodies: CD34 (BB, #343811). CD33 (BioLegend, #303416), CD45 (BioLegend, #304036), CD11 b (BD, #557754), CD15 (BE, #555402), CD16 (BD, #561248), CD66b (Biolegend, #305104). Morphology of the cells was investigated on cytospin slides by Wright-Giemsa staining.

2. Results

[0123] To perform ELANE transcriptional repression using CRISPR/Cas9 D10A nickase, HSPCs of an ELANE-CN patient with a p.A57V mutation were nucleofected with CRISPR/Cas9 D10A RNP and two days later cells were processed for liquid culture differentiation towards neutrophils (FIG. 1A). The same approach was used with healthy volunteers (data not shown).

[0124] Using the double nick strategy in ELANE-CN HSPCs, the inventors observed markedly elevated neutrophil differentiation, as assessed by the percentage of CD45.sup.+CD11b.sup.+CD15.sup.+, CD45.sup.+CD15.sup.+CD16.sup.+ and CD45.sup.+CD16.sup.+CD66b.sup.+ cells (FIGS. 1B, C and 2A, B) compared to ELANE-CN control cells nucleofected with non-targeting CRISPR/Cas9 D10A RNP. No effects were observed with healthy volunteers (data not shown).

[0125] Again, comparing morphological analysis of ELANE-CN with promoter KO to control cells (see above) the strongly improved number of mature granulocytes was confirmed (FIG. 2C, D).

[0126] Similar results were achieved by colony forming unit assay (CFU) showing significantly more granulocytic colony-forming units (CFU-G colonies) in the ELANE promoter KO cells compared to non-targeting CRISPR/Cas9 D10A RNP control (FIG. 2E). Again, effects were observed with healthy volunteers (data not shown).

[0127] The inventors further tested the functionality of in vitro generated ELANE promoter KO neutrophils by testing the reactive oxygen species (ROS) production in response to fMLP. Indeed, ELANE promoter KO cells produced significantly more ROS) in response to fMLP, as compared to control cells with non-targeting CRISPR/Cas9 D10A RNP (FIG. 3A).

[0128] ELANE mRNA expression was reduced by around 5-fold in neutrophils from ELANE promoter KO cells compared to neutrophils from healthy donors (FIG. 3B). Indel's prevalence at targeted site was 87% as assessed by inference of CRISPR edits (ICE) from Sanger Sequencing Trace Data (FIG. 3C), indicating high gene-editing efficiency.

[0129] No effects were observed with healthy volunteers (data not shown).

[0130] The inventors also performed an independent in silica on- and off-target profiling of each sgRNA for up to 4 mismatches using Doench 2016 algorithm of CRISPRitz tool (FIG. 4A, B). The inventors found low off-target effects for each single sgRNAs, indicating very low off-target effects of the combination of both sgRNAs (FIG. 4A, B).

[0131] To further obtain an NGS grade resolution on on-target profile of editing outcome, the inventors used rhAmpSeq CRISPR Analysis System. They found that the editing efficiency was 93%. In FIG. 5A, B, the nucleotide composition across the edited region is depicted along with the frequency of any modification across the edited site.

[0132] To investigate if the inventors' proposed CRISPR/Cas9 strategy may affect granulocytic differentiation, HSPCs of two healthy controls were nucleofected with CRISPR/Cas9 D10A RNP and two days later cells were processed for liquid culture differentiation towards neutrophils. The inventors observed no impairment in neutrophil differentiation of edited cells, as assessed by the percentage of CD45+CD11b+CD15+, CD45+CD15+CD16+ and CD45+CD16+CD66b+ neutrophilic cells that was comparable to healthy control cells nucleofected with non-targeting CRISPR/Cas9 D10A RNP (FIG. 6A, B). While ELANE mRNA expression was reduced significantly in neutrophils from ELANE promoter KO cells, compared to neutrophils from healthy donors nucleofected with non-targeting CRISPR/Cas9 D10A RNP, the inventors observed no changes in mRNA expression levels of two NE related serine proteases, PRTN3 and AZU1 (FIG. 6A, B).

[0133] Therapeutic use of CRISPR/Cas9 in clinic needs a comprehensive knowledge of potential off-target effects to minimize the risk of harmful consequences. Therefore, the inventors performed GUIDE-seq analysis, to be able to have an unbiased genome-wide profiling of off-target cleavage by CRISPR/Cas9 D10A double nickase targeting the ELANE gene promoter. The inventors observed two potential off-target sites, LINC00992-Long Intergenic Non-Protein Coding RNA 992- and Chr9:93703739a noncoding intergenic regionwhich may happen if the RNA guided nuclease had 6 or 7 mismatches with the genome. The GUIDE-seq results highlights the safety profile of ELANE promoter editing approach for clinic (FIG. 7A, B).

3. Conclusion

[0134] Taken together, the inventors established a safe approach for reducing a gene's expression by CRISPR/Cas9 double nick strategy without affecting the gene's coding sequence. This approach was exemplified as feasible using the ELANE gene involved in the development of congenital neutropenia. As demonstrated by examples the approach according to the invention can rescue granulopoiesis in ELANE-CN patients. In healthy individuals the knock-out of the ELANE promoter has no effect on granulopoiesis. Therefore, the invention offers a long-term treatment option replacing the daily G-CSF therapy and decreasing the risk of leukemia development.

[0135] Neutrophil elastase (NE) is a proteolytic enzyme of the neutrophil serine protease (NSP) family, including also cathepsin G (CG), proteinase 3 (PR3) and azurocidin (AZU1). NSPs are stored in cytoplasmic granules, can be secreted into the extra- and peri-cellular space upon cellular activation and considered to be crucially involved in bacterial defense. Elane.sup./ mice have normal neutrophil counts, but there are conflicting results regarding the effect of NE-deficiency on neutrophil extravasation to sites of inflammation, phagocytosis, and neutrophil extracellular traps in mice. NE may or may not be essential for these processes. Papillon-Lefevre Syndrome (PLS) is a human disorder known to cause NE deficiency. This rare autosomal recessive disease is due to loss-of-function mutations in the DPPI gene locus with the loss of the lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI). The activation NSP, including NE, depends on the N-terminal processing activity of DPPI. Therefore, PLS patients exhibit a severe reduction in the activity and stability of all three NSP. Intriguingly, patients with PLS have no defects in their ability to kill bacteria e.g. Staphylococcus aureus or Escherichia coli, suggesting that redundancies in the neutrophil's bactericidal mechanisms negate the necessity for serine proteases for killing common bacteria. Based on these observations, at this juncture, the inventors believe that CRISPR/Cas9 based knockout of ELANE in HSPC of CN patients may restore defective granulopoiesis in CN patients without seriously impairing neutrophil functions.