Somatic haploid human cell line
The invention provides for a somatic fully haploid, karyotypically stable human cell line, e.g. obtainable by targeted deletion of one or more disomic chromosomal regions of a somatic near-haploid human parental cell, a method of producing the same, as well as the use of the cell line for producing isogenic cell variants, comprising genomic mutations at different genomic target sites, and a library of such cell variants.
1. A method of producing a somatic karyotypically stable fully haploid human cell line, comprising:
a) providing a somatic near-haploid human parental cell, wherein the somatic near-haploid human parental cell does not comprise more than 5 chromosomes in two or more copies;
b) identifying a disomic chromosomal region in a genome of the parental cell;
c) deleting the identified disomic chromosomal region using a CRISPR/Cas9 system to obtain a fully haploid cell, wherein the deleted chromosomal region has a length of at least 1 million bp; and
d) expanding the fully haploid cell to obtain the somatic karyotypically stable fully haploid human cell line.
2. The method of claim 1 , wherein the parental cell is derived from a cancer patient.
3. The method of claim 1 , wherein step c) comprises:
i) targeting sites adjacent to the 5′ and 3′ ends of DNA of the chromosomal region, each by a guide RNA (gRNA) including an oligonucleotide sequence that hybridizes with the respective targeted site; and
ii) cleaving DNA at each targeted site upon contact with an RNA-guided endonuclease which catalyzes a DNA double-strand break upon hybridizing with the gRNA, thereby deleting the chromosomal region and obtaining the fully haploid cell.
4. The method of claim 3 , wherein at least one of the gRNAs comprises a sequence selected from the group consisting of SEQ ID 3, SEQ ID 13, SEQ ID 19, and any of SEQ ID 25-47.
5. The method of claim 3 , wherein the RNA-guided endonuclease is selected from the group consisting of Cas9 enzymes originating from any of Streptococcus pyogenes, Streptococcus thermophiles, Neisseria meningitis or Treponema denticola , and functional variants of any of the foregoing, including Cas9 nickases and artificial enzymes.
6. The method of claim 3 , wherein:
A) at least one of the gRNAs comprises a nucleotide sequence chosen from SEQ ID 3, SEQ ID 25, or SEQ ID 26; and the RNA-guided endonuclease comprises an amino acid sequence chosen from SEQ ID 1, SEQ ID 5, SEQ ID 7, SEQ ID 8, or SEQ ID 9; or or
B) at least one of the gRNAs comprises a nucleotide sequence chosen from SEQ ID 13, one of SEQ IDs 27-40; and the RNA-guided endonuclease comprises an amino acid sequence chosen from SEQ ID 10, SEQ ID 15; or
C) at least one of the gRNAs comprises a nucleotide sequence chosen from SEQ ID 19, one of SEQ IDs 41-47; and the RNA-guided endonuclease comprises an amino acid sequence chosen from SEQ ID 16, SEQ ID 21.
7. The method of claim 3 , wherein the DNA double-strand break is proximal to a protospacer associated motif (PAM), and a genomic mutation is obtained by homology-directed repair or by non-homologous end joining.
8. The method of claim 2 , wherein the parental cell is derived from a patient having leukemia or a solid tumor.
9. The method of claim 7 , wherein the DNA double-strand break is 3 bp upstream of the PAM.
10. The method of claim 1 , wherein the deleted chromosomal region has a length of at least 10 million bp.