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US-20260125692-A1 - COUNTER-SELECTION BY INHIBITION OF CONDITIONALLY ESSENTIAL GENES

US20260125692A1US 20260125692 A1US20260125692 A1US 20260125692A1US-20260125692-A1

Abstract

The present invention relates to a method for counter-selection by inhibition of conditionally essential genes

Inventors

  • Steen Troels Joergensen
  • Michael Dolberg Rasmussen

Assignees

  • NOVOZYMES A/S

Dates

Publication Date
20260507
Application Date
20251222
Priority Date
20190625

Claims (20)

  1. 1 - 14 . (canceled)
  2. 15 . A method for inserting at least one polynucleotide of interest into the genome of a host cell, the method comprising the steps of: a) providing a host cell comprising in its genome: i. a polynucleotide encoding a selectable marker comprising a target sequence flanked by a functional PAM sequence for an RNA-guided endonuclease; ii. at least one polynucleotide encoding a gRNA that is at least 80% complementary to and capable of hybridizing to the target sequence; and iii. a polynucleotide encoding a nuclease-null variant of an RNA-guided endonuclease capable of interaction with the gRNA and binding to the target sequence, whereby expression of the selectable marker is repressed; b) transforming said host cell with at least one polynucleotide of interest and capable of inactivating the at least one polynucleotide encoding the gRNA; c) selecting for the trait conferred by the selectable marker; and d) identifying a transformed host cell, wherein the at least one polynucleotide encoding the gRNA has been inactivated by the at least one polynucleotide of interest.
  3. 16 . A method for inserting at least two different polynucleotides of interest into the genome of a host cell, the method comprising the steps of: a) providing a host cell comprising in its genome: i. at least two polynucleotides encoding at least two different selectable markers, each comprising a different target sequence flanked by a functional PAM sequence for an RNA-guided endonuclease; ii. at least two polynucleotides encoding at least two gRNAs that are at least 80% complementary to and capable of hybridizing to the at least two different target sequences; iii. a polynucleotide encoding a nuclease-null variant of an RNA-guided endonuclease capable of interacting with the at least two gRNAs and binding to the at least two different target sequences, whereby expression of the two different selectable markers is repressed; b) transforming said host cell with at least two different polynucleotides of interest, said polynucleotides being capable of inactivating the at least two polynucleotides encoding the at least two gRNAs; and c) selecting for the traits conferred by the at least two different selectable markers; and d) identifying a transformed host cell, wherein the at least two polynucleotides encoding the at least two gRNAs have been inactivated by the at least two different polynucleotides of interest.
  4. 17 . The method according to claim 15 , wherein the at least one polynucleotide of interest encodes an enzyme.
  5. 18 . The method according to claim 15 , wherein the at least one polynucleotide of interest encodes an enzyme selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; most preferably an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and beta-xylosidase.
  6. 19 . The method according to claim 15 , wherein the host cell is a prokaryotic host cell selected from the group consisting of a Bacillus, Streptomyces, Streptococcus , and Lactobacillus cell.
  7. 20 . The method according to claim 15 , wherein the host cell is a Bacillus licheniformis cell.
  8. 21 . The method according to claim 15 , wherein the host cell is a fungal host cell selected from the group consisting of an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , and Trichoderma cell.
  9. 22 . The method according to claim 15 , wherein the host cell is a yeast host cell selected from the group consisting of a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces , and Yarrowia cell.
  10. 23 . The method according to claim 15 , wherein the selectable marker is a positive selection marker, a negative selection marker, a bidirectional marker, or a conditionally essential gene.
  11. 24 . The method according to claim 15 , wherein the selectable marker is selected from the group of genes consisting of cat, erm, tet, amp, spec, kana, neo, dal, lysA, araA, galE, antK, metC, xylA, gntP, glpD, glpF, glpK, glpP, lacA2, hisC, gapA, and aspB.
  12. 25 . The method according to claim 15 , wherein the gRNA comprises a first RNA comprising 20 or more nucleotides that are at least 90% complementary to and capable of hybridizing to the polynucleotide(s) encoding the selectable marker.
  13. 26 . The method according to claim 15 , wherein the RNA-guided endonuclease has a sequence identity of at least 80% to SEQ ID NO: 2
  14. 27 . The method according to claim 15 , wherein the RNA-guided endonuclease has a sequence that comprises or consists of SEQ ID NO: 2.
  15. 28 . The method according to claim 15 , wherein the polynucleotide encoding the RNA-guided endonuclease has a sequence identity of at least 80% to SEQ ID NO: 1.
  16. 29 . The method according to claim 15 , wherein the polynucleotide encoding the RNA-guided endonuclease has a sequence that comprises or consists of SEQ ID NO: 1.
  17. 30 . The method according to claim 15 , wherein the nuclease-null variant of an RNA-guided endonuclease comprises an alteration of an amino acid corresponding to position 877 of SEQ ID NO: 2.
  18. 31 . The method according to claim 30 , wherein the nuclease-null variant of an RNA-guided endonuclease comprises a substitution of aspartic acid for alanine at a position corresponding to position 877 of SEQ ID NO: 2.
  19. 32 . The method according to claim 15 , wherein the PAM sequence is selected from the group consisting of TTTA, TTTT, TTTG, and TTTC.
  20. 33 . The method according to claim 15 , wherein the PAM sequence is TTTC.

Description

REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing in computer-readable form, which is incorporated herein by reference. FIELD OF THE INVENTION The present invention relates to method for counter-selection by inhibition of conditionally essential genes. BACKGROUND OF THE INVENTION The so-called CRISPR genome editing system has been widely used as a tool to modify the genomes of a number of organisms. The power of the CRISPR system lies in its simplicity and its ability to target and edit down to a single base pair in a specific gene of interest. The system relies on CRISPR-associated proteins (Cas), which are RNA-guided endonucleases, as well as so-called guide-RNA (gRNA) molecules that are able to form a complex with the endonuclease and direct the nuclease activity to a particular DNA sequence. The choice of DNA target sequence is made by varying the nucleotide sequence of the gRNA to match the target DNA sequence. When complexed with the gRNA molecule, the endonuclease can recognize and bind its target DNA sequence, forming an endonuclease-gRNA-DNA complex, and create a double-stranded break using its catalytic domain(s). For genome editing purposes, the most widely used CRISPR-associated proteins are those of Class 2, which include Cas9 (Cas type II) derived from Streptococcus pyogenes and Cpf1 (Cas type V) derived from Acidaminococcus or Lachnospiraceae. Another example of an RNA-guided endonuclease is Mad7 isolated from Eubacterium rectale. Although there are some structural similarities between Mad7 and Cpf1, Mad7 is only 31% conserved with Cpf1 from Acidominococcus sp. at the amino acid level. In addition to its use within genome editing, the CRISPR system can also be used to control gene expression. This application, often referred to as CRISPR interference or CRISPRi, allows sequence-specific repression or activation of a gene. CRISPR interference utilizes a catalytically inactive (“dead”) endonuclease variant (e.g., Mad7d) that can be obtained by introducing amino acid mutations in the catalytic domain responsible for endonuclease activity. Upon association with gRNA, the resulting complex retains the ability to bind to the target DNA sequence but cannot introduce any breaks in the DNA strand. As long as the catalytically inactive endonuclease is bound to the target DNA sequence, expression of the target sequence is repressed. By varying the gRNA sequence, one can control the target DNA sequence and thereby regulate the expression of virtually any gene in any organism. Within industrial biotechnology, there is a continued need for robust and effective selection systems suitable for development of optimized production hosts. Given the versatility and precision of the CRISPR technology, it has been speculated that this system could be harnessed for counter-selection purposes. However, attempts of utilizing the CRISPR technology for direct selection have so far been difficult. This is especially true for bacterial host cells, since many prokaryotic organisms are very sensitive to the endonuclease activity of the RNA-guided endonuclease-gRNA complex due to the inefficient repair mechanisms for double-stranded (DS) breaks by non-homologous end-joining (NHEJ) systems that are known from eukaryotes (see, e.g., Su et al., Scientific Reports 2016, 6, 37895; Altenbuchner, Applied and Environmental Microbiology 2016, 82, pp. 5421-5427; Peters et al., Current Opinion in Microbiology 2015, 27, pp. 121-126; Aravind and Koonin, Genome Research 2001, 11, pp. 1365-1374). Moreover, in many cases it is desirable to introduce several copies of a gene or operon (expression cassette) to maximize the yield of a given polypeptide of interest. However, the direct selection using the CRISPR technology will be increasingly difficult if more than one site is targeted for DS breaks in an effort to introduce multiple expression cassettes in one process. Researchers have reported successful integration of a gene of interest (GOI) by homologous recombination (HR) into a gRNA target on chromosome and then introduce endonuclease activity for DS breaks to kill the cells which has retained the original gRNA target sequence. In this way, it is possible to efficiently enrich for cells which have received the GOI. However, the timing of these events of HR and DS activity are very important. RNA-guided endonucleases are typically very active in generating DS breaks and should not be expressed until homologous recombination has occurred and removed the target. SUMMARY OF THE INVENTION The present invention provides means and methods for utilizing the versatility and precision of the CRISPR technology in a selection system suitable for microbial host cells. Thus, in a first aspect, the present invention relates to a method for inserting at least one polynucleotide of interest into the genome of a host cell, the method comprising the steps of: a) providing a host cell comprising in its genome: i. a polynu