WO2024109745A1

WO2024109745A1 - Gene editing systems and methods for treating hbv infection

Info

Publication number: WO2024109745A1
Application number: PCT/CN2023/132968
Authority: WO
Inventors: Zhuoan CHENG; Lijie Wang; Huanyu LI; Peixue Li; Xiaodun MOU
Original assignee: CorrectSequence Therapeutics Co., Ltd
Priority date: 2022-11-21
Filing date: 2023-11-21
Publication date: 2024-05-30
Also published as: WO2024109745A9

Abstract

Provided are gene editing systems and methods for disrupting the human hepatitis B virus (HBV) genes and treating HBV infection. Also provided are polynucleotides, vectors, cells, kits and compositions comprising components of the gene editing systems.

Description

GENE EDITING SYSTEMS AND METHODS FOR TREATING HBV INFECTION

FIELD OF DISCLOSURE

The present disclosure generally relates to gene editing systems and methods for disrupting the human hepatitis B virus (HBV) genes and treating HBV infection. Also disclosed are polynucleotides, vectors, cells, kits, and compositions comprising components of the gene editing systems.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority to and benefits of International Application No. PCT/CN2022/133324, filed November 21, 2022, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted as an XML file entitled “sequence listing. xml” having a size of 927, 747 bytes and created on November 21, 2023. The information contained in the Sequence Listing is incorporated by reference herein.

BACKGROUND

Human hepatitis B virus (HBV) is a hepatotropic, partially double-stranded DNA virus. Following HBV infection, about 5%to 10%of adults and up to 90%of young children fail to produce an immune response adequate to clear the virus, and thus subsequently develop chronic hepatitis B (CHB) , which often progresses to liver cirrhosis, hepatocellular carcinoma, and liver failure. Hepatitis B surface antigen (HBsAg) is a major viral component of the envelope for infectious HBV particles. The loss of HBsAg with undetectable serum HBV DNA is defined as a functional cure. However, current antiviral therapies with nucleoside analogues can inhibit the replication of HBV but can not prevent HBV rebound, thus resulting with only a small proportion of patients achieving a functional cure. Thus, there is a need in the art to find a way to treat HBV infection and prevent HBV rebound.

SUMMARY

The present disclosure provides gene editing systems and methods to disrupt, preferably permanently silence, one or more of the HBV genes. A highly specific base editor, transformer base editor (tBE) , is used to induce efficient and precise gene editing in the human hepatitis B virus (HBV) genome without generating double-stranded breaks, wherein the gene editing can suppress HBV replication and viral protein expression. Multiple combinations of main guide RNA (mgRNA) and helper guide RNA (hgRNA) with high editing efficiency are disclosed, which bind to the HBV genome and the integrated HBV DNA in human genome. The gene editing systems and methods disclosed herein can be used in the treatment of HBV infection and/or hepatitis B in clinical practice.

The tBE systems used herein, when using Cas9 nickase (D10A) , is less toxic to cells than Cas9 nuclease as Cas9 nickase activates a lower level of p53-mediated DDR. Besides, the tBE systems can achieve highly specific and efficient base editing at most sites.

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting a human hepatitis B virus (HBV) S gene and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 1-8.

In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences as set forth in Table 2.

In another aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting a PreS1 region on an HBV S gene and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 9-12.

In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences as set forth in Table 3.

In another aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV P gene on and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 13-30.

In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences as set forth in Table 4.

In another aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV X gene and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 31-32.

In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences as set forth in Table 5.

In another aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 33-38.

In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise the sequences as set forth in Table 6.

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-Agenotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 515-518. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-Agenotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 519. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-Agenotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 520-522. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-B genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 531-534. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-B genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 535. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-B genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 536-538. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-C genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 547-550. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-C genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NOs: 551. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-C genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 552-554. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 563-566. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 567. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 568-570. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-E genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 579-582. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-E genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NOs: 583. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-E genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 584-586. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-F genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 595-598. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-F genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 599. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-F genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 600-602. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-C genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence selected from SEQ ID NOs: 611-613. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-C genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence of SEQ ID NO: 614. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-C genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence selected from SEQ ID NOs: 615-617. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-D genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence selected from SEQ ID NOs: 625-627. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-D genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence of SEQ ID NO: 628. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-D genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence selected from SEQ ID NOs: 629-631. In some embodiments, the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a main crRNA (mcrRNA) and a first tracrRNA, wherein the hgRNA comprises a helper crRNA (hcrRNA) and a second tracrRNA, wherein the mgRNA targets an HBV S gene of HBV-D genotype, wherein the nucleic acid sequence of the mcrRNA comprises a sequence selected from SEQ ID NOs: 639-642. In some embodiments, the nucleic acid sequences of the mcrRNA and the hcrRNA comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a main crRNA (mcrRNA) and a first tracrRNA, wherein the hgRNA comprises a helper crRNA (hcrRNA) and a second tracrRNA, wherein the mgRNA targets an HBV Pre-S1 gene of HBV-D genotype, wherein the nucleic acid sequence of the mcrRNA comprises a sequence of SEQ ID NO: 643. In some embodiments, the nucleic acid sequences of the mcrRNA and the hcrRNA comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a main crRNA (mcrRNA) and a first tracrRNA, wherein the hgRNA comprises a helper crRNA (hcrRNA) and a second tracrRNA, wherein the mgRNA targets an HBV C gene of HBV-D genotype, wherein the nucleic acid sequence of the mcrRNA comprises a sequence selected from SEQ ID NOs: 644-646. In some embodiments, the nucleic acid sequences of the mcrRNA and the hcrRNA comprise respectively:

In some embodiments of the gene editing system comprising the first and second tracrRNA, at least one of the first and second tracrRNA comprises a sequence of SEQ ID NO: 655.

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 656-664. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 689-692. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In some embodiments, the gene editing system induces missense mutation.

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting a NTCP receptor and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 700-714. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In some embodiments, the gene editing system described herein comprises a first mgRNA comprising a first mgRNA spacer targeting a first gene, and a second mgRNA comprising a second mgRNA spacer targeting a second gene, wherein the first gene and the second gene are each independently selected from the group consisting of the HBV S gene, the PreS1 region on the HBV S gene, the HBV P gene, the HBV X gene, and the HBV C gene. In some embodiments, the first gene and the second gene are different.

In some embodiments, the gene editing system disclosed herein comprises (1) the hgRNA comprising a first CRISPR motif, the hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) the mgRNA comprising a second CRISPR motif and the mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein) , or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, and (5) a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif, and wherein the first Cas protein and second Cas protein are the same or different.

In some embodiments, the gene editing system disclosed herein comprises (1) the hgRNA comprising a first CRISPR motif, the hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) the mgRNA comprising a second CRISPR motif and the mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein) , or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif, and (6) a protease, or a polynucleotide encoding the protease, and (7) a nucleobase deaminase inhibitor domain, wherein the first Cas protein and second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is connected to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein optionally by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof.

In some embodiments, the gene editing system disclosed herein comprises (1) the hgRNA comprising a first CRISPR motif, the hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) the mgRNA comprising a second CRISPR motif and the mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein) , or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, and (8) a second fusion protein comprising the protease and a second RNA binding domain, or a polynucleotide encoding the second fusion protein, wherein the first Cas protein and second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is connected to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein optionally by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof, wherein the protease and the second RNA binding domain are optionally connected by a linker, wherein the mgRNA further comprises a second protein-binding motif, and wherein the second RNA binding domain binds to the second protein-binding motif.

In some embodiments, the protease is split into a first protease fragment and a second protease fragment, wherein the first or second protease fragment alone is not able to cleave the cleavage site.

In some embodiments, the gene editing system disclosed herein comprises (1) the hgRNA comprising a first CRISPR motif, the hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) the mgRNA comprising a second CRISPR motif and the mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein) , or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, (8) a second fusion protein comprising the first protease fragment and a second RNA binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA binding domain are optionally connected by a linker, and (9) a third fusion protein comprising the second protease fragment and a third RNA binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA binding domain are optionally connected by a linker, wherein the first Cas protein and second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is connected to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein optionally by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof, wherein the mgRNA further comprises a second protein-binding motif and a third protein-binding motif, wherein the second RNA binding domain binds to the second protein-binding motif, and wherein the third RNA binding domain binds to the third protein-binding motif.

In some embodiments, the gene editing system disclosed herein comprises (1) the hgRNA comprising a first CRISPR motif, the hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) the mgRNA comprising a second CRISPR motif and the mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein) , or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, (8) a second fusion protein comprising the first protease fragment and a second RNA binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA binding domain are optionally connected by a linker, and (9) a third fusion protein comprising the second protease fragment and a third RNA binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA binding domain are optionally connected by a linker, wherein the first Cas protein and second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is connected to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein optionally by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof, wherein the mgRNA further comprises a second protein-binding motif and a third protein-binding motif, wherein the second RNA binding domain binds to the second protein- binding motif, wherein the third RNA binding domain binds to the third protein-binding motif, and wherein the second and third RNA binding domains are the same or different, and the second and third protein-binding motifs are the same or different.

In some embodiments, the gene editing system disclosed herein comprises (1) the hgRNA comprising a first CRISPR motif, the hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) the mgRNA comprising a second CRISPR motif and the mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein) , or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, and (8) a second fusion protein comprising the first protease fragment and a second RNA binding domain, or a polynucleotide encoding the second fusion protein, wherein the first Cas protein and second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is connected to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein optionally by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof, wherein the first protease fragment and the second RNA binding domain are optionally connected by a linker, wherein the mgRNA further comprises a second protein-binding motif, and wherein the second RNA binding domain binds to the second protein-binding motif.

In some embodiments, the protease is a TEV protease, a TuMV protease, a PPV protease, a PVY protease, a ZIKV protease, or a WNV protease.

In some embodiments, the protease cleavage site is a self-cleaving peptide, such as the 2A peptides. “2A peptides” are 18-22 amino-acid-long viral oligopeptides that mediate “cleavage” of polypeptides during translation in eukaryotic cells. The designation “2A” refers to a specific region of the viral genome and different viral 2As have generally been named after the virus they were derived from. The first discovered 2A was F2A (foot-and-mouth disease virus) , after which E2A (equine rhinitis A virus) , P2A (porcine teschovirus-1 2A) , and T2A (thosea asigna virus 2A) were also identified. A few non-limiting examples of 2A peptides are provided in SEQ ID NOs: 219-221.

In some embodiments, the protease is a TEV protease. In some embodiments, the TEV protease comprises a sequence as set forth in SEQ ID NO: 205.

In some embodiments, the first and/or the second TEV protease fragment is not able to cleave the TEV cleavage site on its own. However, in the presence of the remaining portion of the TEV protease, this fragment will be able to effectuate the cleavage. The TEV fragment may be the TEV N-terminal domain (e.g., SEQ ID NO: 206) or the TEV C-terminal domain (e.g., SEQ ID NO: 207) . In some embodiments, the first TEV protease fragment comprises a sequence of SEQ ID NO: 206. In some embodiments, the first TEV protease fragment comprises a sequence of SEQ ID NO: 207.

In some embodiments, the nucleobase deaminase inhibitor is an inhibitory domain of a nucleobase deaminase.

In some embodiments, the nucleobase deaminase inhibitor is an inhibitory domain of a cytidine deaminase. In some embodiments, the nucleobase deaminase inhibitor is the mouse APOBEC3 cytidine deaminase domain 2 (mA3-CDA2, SEQ ID NO: 222) . In some embodiments, the nucleobase deaminase inhibitor is the human APOBEC3B cytidine deaminase domain 1 (hA3B-CDA1, SEQ ID NO: 223) .

In some embodiments, the inhibitory domain of a cytidine deaminase comprises an amino acid sequence as set forth in SEQ ID NO: 222 or SEQ ID NO: 223.

In some embodiments, the nucleotide deaminase is a cytidine deaminase. In some embodiments, the nucleotide deaminase is a cytidine deaminase comprising an amino acid sequence of SEQ ID NO: 224. In some embodiments, the nucleotide deaminase is a cytidine deaminase comprising an amino acid sequence of SEQ ID NO: 225.

In some embodiments, the cytidine deaminase is selected from the group consisting of APOBEC3B (A3B) , APOBEC3C (A3C) , APOBEC3D (A3D) , APOBEC3F (A3F) , APOBEC3G (A3G) , APOBEC3H (A3H) , APOBECI (Al) , APOBEC3 (A3) , APOBEC2 (A2) , APOBEC4 (A4) , and AICDA (AID) .

In some embodiments, the cytidine deaminase is a human or mouse cytidine deaminase.

In some embodiments, the catalytic domain of the cytidine deaminase is a mouse A3 cytidine deaminase domain 1 (CDAl) or human A3B cytidine deaminase domain 2 (CDA2) .

In some embodiments, the nucleotide deaminase is an adenosine deaminase.

In some embodiments, the adenosine deaminase is selected from the group consisting of tRNA-specific adenosine deaminase (TadA) , adenosine deaminase tRNA specific 1 (ADAT1) , adenosine deaminase tRNA specific 2 (ADAT2) , adenosine deaminase tRNA specific 3 (ADAT3) , adenosine deaminase RNA specific B1 (ADARB1) , adenosine deaminase RNA specific B2 (ADARB2) , adenosine monophosphate deaminase 1 (AMPD1) , adenosine monophosphate deaminase 2 (AMPD2) , adenosine monophosphate deaminase 3 (AMPD3) , adenosine deaminase (ADA) , adenosine deaminase 2 (ADA2) , adenosine deaminase like (ADAL) , adenosine deaminase domain containing 1 (ADAD1) , adenosine deaminase domain containing 2 (ADAD2) , and adenosine deaminase RNA specific (ADAR) .

In some embodiments, the first fusion protein further comprises an uracil glycosylase inhibitor (UGI) .

In some embodiments, the first fusion protein further comprises a nuclear localization sequence (NLS) .

In some embodiments, a peptide linker is optionally provided between each of the fragments in any of the fusion proteins. In some embodiments, the peptide linker has from 1 to 100 amino acid residues (or 3-20, 4-15, without limitation) . In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%or 90%of the amino acid residues of peptide linker are amino acid residues selected from the group consisting of alanine, glycine, cysteine, and serine.

In some embodiments, the Cas protein is a SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpfl, LbCpfl, FnCpfl, VQR Cas9, EQR Cas9, VRER Cas9, Cas9-NG, xCas9, eCas9, SpCas9-HF1, HypaCas9, HiFiCas9, sniper-Cas9, SpG, SpRY, KKH SaCas9, CjCas9, Cas9-NRRH, Cas9-NRCH, Cas9-NRTH, SsCpfl, PcCpfl, BpCpfl, LiCpfl, PmCpfl, Lb2Cpf1, PbCpfl, PbCpfl, PeCpf1, PdCpf1, MbCpf1, EeCpf1, CmtCpf1, BsCpfl, BhCasl2b, AkCasl2b, BsCasl2b, AmCasl2b, AaCasl2b, RfxCasl3d, LwaCasl3a, PspCasl3b, PguCasl3b, and RanCasl3b.

In some embodiments, the first protein-binding RNA motif and the first RNA binding domain, the second protein-binding RNA motif and the second RNA binding domain, and the third protein-binding RNA motif and the third RNA binding domain, are each independently selected from the group consisting of a MS2 phage operator stem-loop and MS2 coat protein (MCP) or an RNA-binding section thereof; a boxB and N22p or an RNA-binding section thereof; a telomerase Ku binding motif and Ku protein or an RNA-binding section thereof; a telomerase Sm7 binding motif and Sm7 protein or an RNA-binding section thereof; a PP7 phage operator stem-loop and PP7 coat protein (PCP) or an RNA-binding section thereof; a SfMu phage Com stem-loop and Com RNA binding protein or an RNA-binding section thereof; and a non-natural RNA aptamer and corresponding aptamer ligand or an RNA-binding section thereof.

In some embodiments of the gene editing system described herein, the mgRNA and/or the hgRNA comprises a dual-RNA structure. In some embodiments, the dual-RNA structure is formed by a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) , wherein the crRNA comprises the spacer. In some embodiments, the mgRNA comprises a mcrRNA and a first tracrRNA, and the mcrRNA comprises the mgRNA spacer, wherein the hgRNA comprises a hcrRNA and a second tracrRNA, and the hcrRNA comprises the hgRNA spacer, and wherein the first tracrRNA and the second tracrRNA are same or different.

In some embodiments, the mcrRNA and the hcrRNA are

SEQ ID NO. 639 and SEQ ID NO: 647, respectively; or

SEQ ID NO. 640 and SEQ ID NO: 648, respectively; or

SEQ ID NO. 641 and SEQ ID NO: 649, respectively; or

SEQ ID NO. 642 and SEQ ID NO: 650, respectively; or

SEQ ID NO. 643 and SEQ ID NO: 651, respectively; or

SEQ ID NO. 644 and SEQ ID NO: 652, respectively; or

SEQ ID NO. 645 and SEQ ID NO: 653, respectively; or

SEQ ID NO. 646 and SEQ ID NO: 654, respectively.

In some embodiments, the tracrRNA is SEQ ID NO: 655.

In another aspect, the present disclosure provides a polynucleotide encoding the hgRNA and/or the mgRNA disclosed in at least one of the gene editing systems herein.

In another aspect, the present disclosure provides a polynucleotide encoding all components except the first and the second Cas protein in the gene editing system disclosed herein.

In another aspect, the present disclosure provides a polynucleotide encoding all components in the gene editing system disclosed herein.

In another aspect, the present disclosure provides a kit comprising a polynucleotide encoding all components except the first and the second Cas protein in the gene editing system disclosed herein, and a polynucleotide encoding the first and/or second Cas protein in the gene editing system disclosed herein. In some embodiments, the first and the second Cas proteins are the same Cas protein.

In another aspect, the present disclosure provides a vector comprising the polynucleotide encoding the hgRNA and/or the mgRNA disclosed herein.

In another aspect, the present disclosure provides a vector comprising the polynucleotide disclosed herein.

In another aspect, the present disclosure provides a vector comprising the polynucleotide encoding all components except the first and the second Cas protein in the gene editing system disclosed herein.

In another aspect, the present disclosure provides a vector comprising the polynucleotide encoding all components in the gene editing system disclosed herein.

In some embodiments, the vector is a plasmid or a viral vector.

In some embodiments, the vector is a polycistronic vector.

In another aspect, the present disclosure provides a kit comprising the vector disclosed above, and a vector comprising the polynucleotide encoding the first and/or second Cas protein in the gene editing system disclosed herein.

In another aspect, the present disclosure provides a cell comprising any one or more of the gene editing systems disclosed herein.

In another aspect, the present disclosure provides a cell comprising the polynucleotide disclosed herein. In some embodiments, the cell further comprises a polynucleotide encoding the first and/or second Cas protein in the gene editing system disclosed herein.

In another aspect, the present disclosure provides a cell comprising the vector disclosed herein. In some embodiments, the cell further comprises a vector comprising a polynucleotide encoding the first and/or second Cas protein in the gene editing system disclosed herein.

In another aspect, the present disclosure provides a cell comprising the components of the kit disclosed herein.

In some embodiments, the cell is infected by an HBV.

In some embodiments, the cell comprises an HBV covalently closed circular DNA (cccDNA) .

In some embodiments, the cell comprises an HBV integrated DNA.

In some embodiments, the cell is a liver cell. In some embodiments, the cell is a hepatocyte.

In another aspect, the present disclosure provides a composition comprising the gene editing system disclosed herein.

In another aspect, the present disclosure provides a composition comprising the cell disclosed herein.

In another aspect, the present disclosure provides a method for disrupting an HBV gene in a cell, comprising introducing into the cell the gene editing system disclosed herein. In some embodiments, the present disclosure provides a method for disrupting an HBV S gene comprising introducing into the cell the gene editing system disclosed herein, wherein the combination of mgRNA and hgRNA are selected from Table 2 or Table 3. In some embodiments, the present disclosure provides a method for disrupting an HBV P gene comprising introducing into the cell the gene editing system disclosed herein, wherein the combination of mgRNA and hgRNA are selected from Table 4. In some embodiments, the present disclosure provides a method for disrupting an HBV X gene comprising introducing into the cell the gene editing system disclosed herein, wherein the combination of mgRNA and hgRNA are selected from Table 5. In some embodiments, the present disclosure provides a method for disrupting an HBV C gene comprising introducing into the cell the gene editing system disclosed herein, wherein the combination of mgRNA and hgRNA are selected from Table 6. Any two or more of these methods can be used in combination.

In another aspect, the present disclosure provides a method for treating HBV infection in a subject, comprising disrupting an HBV gene in a cell of the subject with any one or more of the methods disclosed herein.

In another aspect, the present disclosure provides a method for treating or preventing chronic hepatitis B (CHB) , liver cirrhosis, hepatocellular carcinoma, and/or liver failure, using any one or more of the gene editing systems disclosed herein.

In some embodiments, the cell is infected by HBV. In some embodiments, the cell comprises an HBV cccDNA. In some embodiments, the cell comprises an HBV integrated DNA. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a hepatocyte.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 illustrates exemplary base editors that can be used in the gene editing systems disclosed here. The various versions of base editors are denoted as V1, V2, V3, V4, and V5, with constructs denoted as tBE-V1-rA1, tBE-V2-rA1, tBE-V3-rA1, tBE-V4-rA1, tBE-V5-rA1, and tBE-V5-mA3. Fig. 1A shows schematic diagrams illustrating the construction and development of various versions of base editors. Fig. 1B shows interactions of molecular components in different versions of the base editors. Base editors of V2 to V5 illustrate different strategies to cleave mA3dCDI off. The dCDI domain could be cleaved off from APOBEC through a two-component interaction of the TEV site and a free TEV protease (V2) , a N22p-fused TEV protease (V3) , or a TEV protease reconstituted by an mgRNA-boxB (V4) . In the version 5 (V5) of the base editor, the dCDI is cleaved off from APOBEC through a three-component interaction of TEV site, TEVn, and N22p-TEVc.

Fig. 2 shows editing efficiencies induced by tBE with the pairs of mgRNA-HBV-S-1～4 and their hgRNAs targeting HBV-Sgene. Fig. 2A is a schematic diagram illustrating the co-transfection of mgRNA-HBV-S-1～4 and their different hgRNA-HBV-S-U1～3 with tBE-V5-mA3 and nCas9. Fig. 2B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites. Fig. 2C shows editing frequency at each target site calculated by EditR analysis.

Fig. 3 shows editing efficiencies induced by tBE with the pairs of mgRNA-HBV-S-5～7 and their hgRNAs targeting HBV-Sgene. Fig. 3A is a schematic diagram illustrating the co-transfection of mgRNA-HBV-S-5～7 and their different hgRNA-HBV-S-U1～3 with tBE-V5-mA3 and nCas9. Fig. 3B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites. Fig. 3C shows editing frequency at each target site calculated by EditR analysis.

Fig. 4 shows editing efficiencies induced by tBE with the pairs of mgRNA-HBV-PreS1-3-4 and their hgRNAs targeting HBV-PreS1 gene. Fig. 4A is a schematic diagram illustrating the co-transfection of mgRNA-HBV-PreS1-3-4 and their different hgRNA-HBV-PreS1-U1～3 with tBE-V5-mA3 and nCas9. Fig. 4B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites. Fig. 4C shows editing frequency at each target site calculated by EditR analysis.

Fig. 5 shows editing efficiencies induced by tBE with the pairs of mgRNA-HBV-P-4～7 and their hgRNAs targeting HBV-P gene. Fig. 5A is a schematic diagram illustrating the co-transfection of mgRNA-HBV-P-4～7 and their different hgRNA-HBV-P-U1～2 with tBE-V5-mA3 and nCas9. Fig. 5B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites. Fig. 5C shows editing frequency at each target site calculated by EditR analysis.

Fig. 6 shows editing efficiencies induced by tBE with the pairs of mgRNA-HBV-P-8～12 and their hgRNAs targeting HBV-P gene. Fig. 6A is a schematic diagram illustrating the co-transfection of mgRNA-HBV-P-8～12 and their different hgRNA-HBV-P-U1～2 with tBE-V5-mA3 and nCas9. Fig. 6B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites. Fig. 6C shows editing frequency at each target site calculated by EditR analysis.

Fig. 7 shows editing efficiencies induced by tBE with the pairs of mgRNA-HBV-P-13, 15, 17, 19 and their hgRNAs targeting HBV-P gene. Fig. 7A is a schematic diagram illustrating the co-transfection of mgRNA-HBV-P-13, 15, 17, 19 and their different hgRNA-HBV-P-U1～3 with tBE-V5-mA3 and nCas9. Fig. 7B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites. Fig. 7C shows editing frequency at each target site calculated by EditR analysis.

Fig. 8 shows editing efficiencies induced by tBE with the pairs of mgRNA-HBV-C-1～4 and their hgRNAs targeting HBV-C gene. Fig. 8A is a schematic diagram illustrating the co-transfection of mgRNA-HBV-C-1～4 and their different hgRNA-HBV-C-U1～3 with tBE-V5-mA3 and nCas9. Fig. 8B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites. Fig. 8C shows editing frequency at each target site calculated by EditR analysis.

Fig. 9 shows editing efficiencies induced by tBE with the pairs of mgRNA-HBV-C-5 and its hgRNAs targeting HBV-C gene. Fig. 9A is a schematic diagram illustrating the co-transfection of mgRNA-HBV-C-5 and its different hgRNA-HBV-C-U1～2 with tBE-V5-mA3 and nCas9. Fig. 9B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites. Fig. 9C shows editing frequency at each target site calculated by EditR analysis.

Fig. 10 is an illustration of the HBV genome. Fig. 10A shows the genomic organization, RNA transcripts, and gene products with several key regulatory elements. Fig. 10B shows the transcription start sites of various HBV transcripts and the proteins they encode.

Fig. 11 shows editing efficiencies induced by tBE with the pairs of mgRNAs and its hgRNAs targeting HBV transcriptional elements S or P or X or C of HBV-C genotype for functional screen in HBV-C-S/P/X/C stably transfected Hek293FT cell. Editing efficiency at each target is calculated by EditR analysis by at least two independent experiments.

Fig. 12 shows editing efficiencies induced by tBE with the pairs of mgRNA and its hgRNAs targeting HBV gene in HBV-A or B or C or D or E or F genotype in HBV stably transfected HepG2 cell.

Fig. 13 shows editing efficiencies induced by a V5-LigoRNA-based editing system in an RNA electroporation delivery system in HepG2 cell. Fig. 13A is a schematic diagram illustrating co-transfection of pairs of crRNA-HBV-S/C and tracrRNAs with mRNA of tBE-V5-mA3 and nCas9. Fig. 13B shows editing efficiencies induced by tracrRNAs with its pairs of crRNA-HBV-S/C in HBV-D genotype stably transfected HepG2 cell.

Fig. 14 shows effective HBsAg and HBeAg reduction with the indicated guide RNA in HBV-C genotype stably transfected HepG2 cell. Fig. 14A shows editing efficiency induced by indicated pairs of mgRNA and its hgRNA combined with tBE mRNA at indicated sites with HBV-C genotype stably transfected HepG2 cell. Fig. 14B shows HBsAg and HBeAg reduced by indicated pairs of mgRNA and its hgRNA combined with tBE mRNA. Fig. 14C is a schematic diagram illustrating co-transfection of mgRNA and corresponding hgRNA with mRNA of tBE-V5-mA3 and nCas9.

Fig. 15 shows effective HBsAg and HBeAg reduction with the indicated guide RNA in HBV-D genotype stably transfected HepG2 cell. Fig. 15A shows editing efficiency induced by indicated pairs of mgRNA and its hgRNA combined with tBE mRNA at indicated sites with HBV-D genotype stably transfected HepG2 cell Fig. 15B shows HBsAg and HBeAg reduced by indicated pairs of mgRNA and its hgRNA combined with tBE mRNA. Fig. 15C is a schematic diagram illustrating co-transfection of mgRNA and corresponding hgRNA with mRNA of tBE-V5-mA3 and nCas9.

Fig. 16 shows effective HBsAg reduction with the indicated guide RNA in PLC cell. Fig. 16A shows editing efficiency induced by indicated pairs of mgRNA and its hgRNA combined with tBE mRNA at indicated sites with PLC cell. Fig. 16B shows HBsAg reduced by indicated pairs of mgRNA and its hgRNA combined with tBE mRNA. Fig. 16C is a schematic diagram illustrating co-transfection of mgRNA and corresponding hgRNA with mRNA of tBE-V5-mA3 and nCas9.

Fig. 17 shows effective HBsAg and HBeAg reduction with the indicated guide RNA in HepG2.2.15. Fig. 17A shows editing efficiency induced by indicated pairs of mgRNA and its hgRNA combined with tBE mRNA at indicated sites in HepG2.2.15. Fig. 18B shows HBsAg and HBeAg reduced by indicated pairs of mgRNA and its hgRNA combined with tBE mRNA. Fig. 17C is a schematic diagram illustrating co-transfection of mgRNA and corresponding hgRNA with mRNA of tBE-V5-mA3 and nCas9.

Fig. 18 shows effective HBsAg and HbeAg reduction with multiplex gRNAs in HBV-C genotype stably transfected HepG2. Fig. 18A shows editing efficiency induced by indicated multiplex pairs of mgRNA and its hgRNA combined with tBE mRNA at indicated sites in HBV-C genotype stably transfected HepG2 cell. Fig. 18B shows HbsAg and HbeAg reduced by multiplex indicated pairs of mgRNA and its hgRNA combined with tBE mRNA. Fig. 18C is a schematic diagram illustrating co-transfection of mgRNA and corresponding hgRNA with mRNA of tBE-V5-mA3 and nCas9.

Fig. 19 shows effective HBsAg and HbeAg reduction with multiplexing gRNA-S-TGG-4+gRNA-C-TGG-1 in HepG2.2.15. Fig. 19A shows editing efficiency induced by gRNA-S-TGG-4 and gRNA-C-TGG-1 combined with tBE mRNA at indicated sites in HepG2.2.15. Fig. 19B shows HbsAg and HbeAg reduced by indicated pairs of mgRNA and its hgRNA combined with tBE mRNA. Fig. 19C is a schematic diagram illustrating co-transfection of mgRNA and corresponding hgRNA with mRNA of tBE-V5-mA3 and nCas9.

Fig. 20 shows editing efficiencies induced by tBE with the pairs of mgRNA-NTCP-1～5 and its hgRNAs targeting NTCP gene. Fig. 20A is a schematic diagram illustrating the co-transfection of mgRNA-NTCP-1, 2, 4, 5 and its different hgRNAs with tBE-V5-mA3 and nCas9/nCas9-SpG. Fig. 20B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites.

Fig. 21 shows editing efficiencies induced by tBE with the pairs of mgRNA-NTCP-11～15 and its hgRNAs targeting NTCP gene. Fig. 21A is a schematic diagram illustrating the co-transfection of mgRNA-NTCP-11～15 and its different hgRNA-NTCP-11～15 with tBE-V5-mA3 and nCas9/nCas9-SpG. Fig. 21B shows editing efficiency induced by tBE-V5-mA3 with indicated pairs of mgRNA/hgRNA at indicated sites.

DETAILED DESCRIPTION

Definition

In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings generally understood by a person skilled in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present disclosure, the preferred methods and materials are described herein. Accordingly, the terms defined herein are more fully described by reference to the Specification as a whole.

As used herein, the singular terms “a, ” “an, ” and “the” include the plural reference unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ( “or” ) . Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

Unless the context requires otherwise, the terms “comprise, ” “comprises, ” and “comprising, ” or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.

Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively. A number “n” , when used in the context of an amino acid sequence, refers to the n^th amino acid in the amino acid sequence counting from the amino end. For example, “amino acid 15” refers to the 15^th amino acid in a certain amino acid sequence. For example, “R15” refers to the 15^th amino acid, which is an arginine (R) , in a certain amino acid sequence.

It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context in which they are used by those skilled in the art.

As used herein, the terms “percent identity” and “%identity, ” as applied to nucleic acid or polynucleotide sequences, refer to the percentage of residue matches between at least two nucleic acid or polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.

Percent identity between nucleic acid or polynucleotide sequences may be determined using a suite of commonly used and freely available sequence comparison algorithms provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215: 403-410) , which is available from several sources, including the NCBI, Bethesda, Md., and on the Internet at http: //www. ncbi. nlm. nih. gov/BLAST/.

Nucleic acid or polynucleotide sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res 19: 5081; Ohtsuka et al. (1985) J Biol Chem 260: 2605-2608; Cassol et al. (1992) ; Rossolini et al. (1994) Mol Cell Probes 8: 91-98) . The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. The term nucleic acid is used interchangeably with polynucleotide, and (in appropriate contexts) gene, cDNA, and mRNA encoded by a gene.

As used herein, “percent (%) amino acid sequence identity” with respect to a peptide, polypeptide or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in another peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent amino acid sequence identity in the current disclosure is measured using BLAST software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

An amino acid substitution refers to the replacement of one amino acid in a polypeptide with another amino acid. Amino acid substitutions can be conservative or non-conservative substitutions. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into a protein of interest and the products screened for a desired activity, for example, retained/improved biological activity.

Table 1 Exemplary Substitutions

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides, ” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds) . The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, “peptides, ” “protein” , or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide, ” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

As used herein, the term “encode” or “encoding” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

A “guide RNA” (gRNA) , refers to a synthetic or expressed RNA sequence that comprises a CRISPR binding motif and a spacer. In some embodiments, the guide RNA is a single guide RNA (e.g., sgRNA, hsgRNA) . In some embodiments, the guide RNA is a dual-RNA structure. In some embodiments, the guide RNA is a dual-RNA structure formed by a ligand-bound CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) . In some embodiments, the guide RNA is a LigoRNA. A “spacer” is a DNA-targeting motif, which is a sequence that is complementary to a target specific DNA region. In some embodiments, the guide RNA is a crRNA-tracrRNA dual RNA structure, and the crRNA comprises the spacer. The CRISPR binding motif of a guide RNA can bind to a Cas enzyme and DNA-targeting motif of the gRNA can guide the complex to a specific target location on a DNA. In some embodiments, the guide RNA is a crRNA-tracrRNA dual RNA structure, and the base-pair structure formed by the crRNA and the tracrRNA comprises the CRISPR binding motif. A guide RNA may further comprise one or more protein-binding motifs.

As used herein, a “fusion protein” is a protein comprising at least two domains that are encoded by separate genes that have been joined a single polypeptide. For example, a fusion protein can comprise two domains that are encoded by separate genes that have been joined so that they are transcribed and translated as a single unit, producing a single polypeptide. In some embodiments, the at least two domains are fused together directly. In some embodiments, the domains are connected by one or more linkers.

The term “genetic modification” and its grammatical equivalents as used herein can refer to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome. For example, genetic modification can refer to alterations, additions, and/or deletion of genes or portions of genes or other nucleic acid sequences. A genetically modified cell can also refer to a cell with an added, deleted, and/or altered gene or portion of a gene. A genetically modified cell can also refer to a cell with an added nucleic acid sequence that is not a gene or gene portion. Genetic modifications include, for example, both transient knock-in or knock-down mechanisms, and mechanisms that result in permanent knock-in, knock-down, or knock-out of target genes or portions of genes or nucleic acid sequences. Genetic modifications include, for example, both transient knock-in and mechanisms that result in permanent knock-in of nucleic acids sequences. Genetic modifications also include, for example, reduced or increased transcription, reduced or increased mRNA stability, reduced or increased translation, and reduced or increased protein stability.

As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells.

The term “subject” means any animal such as a mammal, e.g., a human.

As used herein, the term “treat, ” “treating, ” or “treatment” refers to ameliorating a disease or disorder, e.g., slowing or arresting or reducing the development of the disease or disorder or reducing at least one of the clinical symptoms thereof. For example, in some embodiments, ameliorating a disease or disorder can include obtaining a beneficial or desired clinical result that includes, but is not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting or eliminating the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total) .

For the purpose of this disclosure, the term “HBV” includes all genotypes of the hepatitis B virus, unless specifies otherwise.

Human Hepatitis B Virus

Human hepatitis B virus (HBV) is a hepatotropic virus. Following HBV infection, 5%–10%of adults and up to 90%of young children fail to produce an immune response adequate to clear the virus, and thus subsequently develop chronic hepatitis B (CHB) , which often progresses to liver cirrhosis, hepatocellular carcinoma, and liver failure. At least eight distinct genotypes (Ato H) of HBV have been identified with over 8%genetic divergence among the viral genomes. Genotype B and C are both common in Asia and genotype C is the main genotype in China.

HBV is the prototypic member of the Hepadnavirdae family, composed of enveloped viruses that contain about 3.2 kbp relaxed circular double-stranded DNA (dsDNA) genomes encapsidated within virally-encoded capsids. Its small genome exhibits high informational density with extensively overlapping open reading frames (ORFs) , with every base coding for at least one ORF. The genome contains four genes, which result in different RNAs with a common poly-Asite, coding for seven different proteins, including the structural proteins, HBsAg (of which there are three forms: large, medium, and small) and HBV core antigen (HBcAg) ; HBV e antigen (HBeAg) , a processed and secreted form of the gene product of the preCore/Core ORF; the HBV polymerase (pol) ; and the transcriptional transactivator HBV X protein (HBx) , which controls HBV transcription from cccDNA. (see Fig. 10)

The four genes included in the HBV genome are S gene, C gene, P gene, and X gene (for example, in HBV genotype C, the four genes have a sequence of SEQ ID NOs: 328-331, respectively) . The S gene, which encodes the viral surface envelope proteins, the HBsAg, has one long open reading frame containing three in frame "start" (ATG) codons that divide the gene into three sections, pre-S1 (for example, in HBV genotype C, the pre-S1 region is SEQ ID NO: 332) , pre-S2, and S. Because of the multiple start codons, polypeptides of three different sizes called large, middle, and small (pre-S1 + pre-S2 + S, pre-S2 + S, or S) are produced.

Similarly, the C gene also has multiple in frame start codons. The core protein is coded by gene C (HBcAg) , and its start codon is preceded by an upstream in-frame AUG start codon from which the pre-core protein is produced. HBeAg is produced by proteolytic processing of the pre-core protein.

Gene P encodes the DNA polymerase. The polymerase (pol) is a large protein (about 800 amino acids) which is functionally divided into three domains: the terminal protein domain, which is involved in encapsidation and initiation of minus-strand synthesis; the reverse transcriptase (RT) domain, which catalyzes genome synthesis; and the ribonuclease H domain, which degrades pregenomic RNA and facilitates replication.

Gene X encodes a 16.5-kd protein, HBxAg, which has multiple functions, including signal transduction, transcriptional activation, DNA repair, and inhibition of protein degradation. HBxAg is necessary for productive HBV infection in vivo and may contribute to the oncogenic potential of HBV. (Tong, Shuping, and Peter Revill. "Overview of hepatitis B viral replication and genetic variability. " Journal of hepatology 64.1 (2016) : S4-S16; Liang TJ. Hepatitis B: the virus and disease. Hepatology. 2009 May; 49 (5 Suppl) : S13-21. doi: 10. 1002/hep. 22881. PMID: 19399811; PMCID: PMC2809016)

The HBV replication cycle starts with attachment and entry into cells, for example, hepatocytes, the main cell type of the liver. HBV initially attaches via low-specificity interactions between HBsAg within the virus envelope and heparan sulphate proteoglycans on the surface of hepatocytes. A high specificity interaction then occurs between the N-terminal 75 amino acids of the preS1-domain of the large HBsAg and sodium taurocholate cotransporting polypeptide (NTCP) , a hepatocyte‐specific bile salt transporter and cellular receptor for HBV. NTCP, a sodium taurocholate co-transporting polypeptide, a bile acid transporter predominantly expressed in the liver, has been identified as a functional receptor for HBV and hepatitis delta virus (HDV) .

After receptor-mediated entry of the virion, the nucleocapsid containing the relaxed circular DNA (rcDNA) genome is released into the cytoplasm and transported to the nucleus. The liberated rcDNA is converted into covalently closed circular DNA (cccDNA) , the stable episomal transcriptional template for HBV messenger RNAs (mRNAs) . Both subgenomic mRNAs encoding the structural and regulatory proteins, and greater-than-genome-length pregenomic RNA (pgRNA) are transcribed from cccDNA. pgRNA, along with the viral polymerase, is encapsidated into viral capsids. Reverse transcription of the pgRNA occurs within the nucleocapsid, resulting in rcDNA or double-stranded linear DNA (dslDNA) forms. (Tu T, Budzinska MA, Shackel NA, Urban S. HBV DNA Integration: Molecular Mechanisms and Clinical Implications. Viruses. 2017 Apr 10; 9 (4) : 75. doi: 10.3390/v9040075. PMID: 28394272; PMCID: PMC5408681)

HBV DNA integration occurs throughout the host genome at dsDNA breaks, with terminal deletions of up to 200 bp from the integrated HBV DNA being common. DNA integration is a process in which double-stranded viral DNA is inserted into the host cell genome, resulting in an integrated DNA. An HBV integrated DNA refers to a piece of host cell genomic DNA that is inserted with HBV DNA. HBV DNA integration has been generally reported in both hepatocellular carcinoma (HCC) and cirrhotic patients with long-term chronic hepatitis B (CHB) , and recently also prior to histologically observable liver damage in CHB patients.

The present disclosure provides gene editing systems and methods to disrupt, preferably permanently silence, HBV genes in cccDNA and integrated HBV DNA.

Gene Editing Systems

The safety and efficiency of gene editing tools are of great importance in clinical applications. Previous studies have reported that the DSBs induced by Cas9 nuclease can activate a p53-mediated DDR pathway and then lead to cell death. Moreover, APOBEC/AID family members can trigger C-to-T base substitutions in single-stranded DNA (ssDNA) regions, which are formed randomly during various cellular processes including DNA replication, repair and transcription. Thus, the specificity of previous base editing systems is compromised, limiting the applications of BEs for therapeutic purposes.

The present disclosure provides a newly developed base editing system, transformer base editor (tBE) , which can specifically edit cytosine in target regions with no observable off-target mutations.

In some embodiments, the transformer base editor (tBE) system contains a cytidine deaminase inhibitor (dCDI) domain and a split-TEV protease. Thus, tBE remains inactive at off-target sites with a cleavable fusion of dCDI domain and eliminates unintended off-target mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise editing. Specifically, tBE uses one mgRNA (normally 20 nt) to bind at the target genomic site and one helper guide RNA (hgRNA, normally 10 to 20 nt) to bind at a nearby region (preferably upstream to the target genomic site) . The binding of the two gRNAs can guide the components of tBE system to correctly assemble at the target genomic site for base editing. tBE can specifically edit cytosine in target regions with no observable off-target mutations, e.g., inducing a premature stop codon to repress HBV protein expression.

In this disclosure, the tBE system is used to disrupt an HBV gene in cccDNA and/or integrated HBV DNA, which leads to suppression of the expression of the viral core protein, surface envelope proteins, polymerase, and/or HBxAg. The base editors and base editing methods described in this disclosure could be applied to perform high-specificity and high-efficiency base editing in the genome of HBV and various other viruses.

In some embodiments, the present disclosure provides tBE systems and mgRNA/hgRNA combinations used in the tBE systems that target the S gene, PreS1 region of the S gene, the P gene, the X gene, and/or the C gene of HBV.

In some embodiments, the tBE is any one of the base editors described in WO2020156575A1, incorporated herein by reference in its entirety. For instance, the tBE can be any base editor as illustrated in Fig. 1.

In some embodiments, a base editor as used herein is a cytosine base editor (CBE) , which comprises a combination of a CRISPR system and cytidine deaminase. A CBE effectuates a programmable cytosine to thymine (C-to-T) substitution. Because the base editing process does not depend on the generation of DNA double strand break (DSB) , unwanted nucleotide insertions/deletions (indels) or DNA damage responses (DDRs) can be largely avoided.

In some embodiments, the gene editing system disclosed herein disrupts the targe gene by generating stop codons or destroy splicing sites in the target gene.

In some embodiments, the gene editing system disclosed herein induces C-to-T base editing in the codons of CAA (Gln) , CAG (Gln) , or CGA (Arg) in the target gene to create a TAA, TAG, or TGA stop codon.

In some embodiments, the gene editing system disclosed herein induces G-to-Abase editing in the codons of TGG (Trp, C-to-T on the opposite strand) to create a TAA, TAG, or TGA stop codon.

In some embodiments, the present disclosure provides a gene editing system for disrupting an HBV S gene, wherein the gene editing system comprises a base editor and at least one guide RNA that is capable of binding to the HBV S gene. In some embodiments, a highly specific base editor, transformer base editor (tBE) , is used to induce efficient and precise gene editing at genomic sites for disrupting the HBV S gene. A tBE comprises a combination of main guide RNA (mgRNA) and helper guide RNA (hgRNA) , wherein the mgRNA and hgRNA are capable of binding to the HBV S gene.

In some embodiments, the present disclosure provides a gene editing system for disrupting a PreS1 region of an HBV S gene, wherein the gene editing system comprises a base editor and at least one guide RNA that is capable of binding to the PreS1 region of the HBV S gene. In some embodiments, a highly specific base editor, transformer base editor (tBE) , is used to induce efficient and precise gene editing at genomic sites for disrupting the PreS1 region of the HBV S gene. A tBE comprises a combination of main guide RNA (mgRNA) and helper guide RNA (hgRNA) , wherein the mgRNA and hgRNA are capable of binding to the PreS1 region of the HBV S gene.

In some embodiments, the present disclosure provides a gene editing system for disrupting an HBV P gene, wherein the gene editing system comprises a base editor and at least one guide RNA that is capable of binding to the HBV P gene. In some embodiments, a highly specific base editor, transformer base editor (tBE) , is used to induce efficient and precise gene editing at genomic sites for disrupting the HBV P gene. A tBE comprises a combination of main guide RNA (mgRNA) and helper guide RNA (hgRNA) , wherein the mgRNA and hgRNA are capable of binding to the HBV P gene.

In some embodiments, the present disclosure provides a gene editing system for disrupting an HBV X gene, wherein the gene editing system comprises a base editor and at least one guide RNA that is capable of binding to the HBV X gene. In some embodiments, a highly specific base editor, transformer base editor (tBE) , is used to induce efficient and precise gene editing at genomic sites for disrupting the HBV X gene. A tBE comprises a combination of main guide RNA (mgRNA) and helper guide RNA (hgRNA) , wherein the mgRNA and hgRNA are capable of binding to the HBV X gene.

In some embodiments, the present disclosure provides a gene editing system for disrupting an HBV C gene, wherein the gene editing system comprises a base editor and at least one guide RNA that is capable of binding to the HBV C gene. In some embodiments, a highly specific base editor, transformer base editor (tBE) , is used to induce efficient and precise gene editing at genomic sites for disrupting the HBV C gene. A tBE comprises a combination of main guide RNA (mgRNA) and helper guide RNA (hgRNA) , wherein the mgRNA and hgRNA are capable of binding to the HBV C gene.

Table 2 Combinations of mgRNA and hgRNA (Gene S)

Table 3 Combinations of mgRNA and hgRNA (PreS1 of Gene S)

In another aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV P gene on and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 13-30

Table 4 Combinations of mgRNA and hgRNA (Gene P)

In another aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV X gene and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 31-32

Table 5 Combinations of mgRNA and hgRNA (Gene X)

Table 6 Combinations of mgRNA and hgRNA (Gene C)

In an aspect, the present disclosure provides In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-Agenotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 515-518. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-Agenotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 519. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-Agenotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 520-522. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-B genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 531-534. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In an aspect, the present disclosure provides a gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-E genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 583. In some embodiments, the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:

In some embodiments, the gene editing system further comprises an adenosine deaminase or a functional fragment thereof.

In some embodiments, the gene editing system induces missense mutation. A missense mutation is a mutation that results in a codon that codes for a different amino acid at the mutation site. It is a type of nonsynonymous substitution.

In some embodiments, the gene editing system described herein comprises a first mgRNA comprising a first mgRNA spacer targeting a first gene, and a second mgRNA comprising a second mgRNA spacer targeting a second gene, wherein the first gene and the second gene are each selected from the group consisting of the HBV S gene, the PreS1 region on the HBV S gene, the HBV P gene, the HBV X gene, and the HBV C gene. In some embodiments, the first gene and the second gene are different.

In some embodiments, the gene editing system disclosed herein comprises (1) the hgRNA comprising a first CRISPR motif, the hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA, (2) the mgRNA comprising a second CRISPR motif and the mgRNA spacer, or a DNA polynucleotide encoding the mgRNA, (3) a first CRISPR-associated protein (Cas protein) , or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif, (4) a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif, (5) a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif, (6) a protease, or a polynucleotide encoding the protease, (7) a nucleobase deaminase inhibitor domain, (8) a second fusion protein comprising the first protease fragment and a second RNA binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA binding domain are optionally connected by a linker, and (9) a third fusion protein comprising the second protease fragment and a third RNA binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA binding domain are optionally connected by a linker, wherein the first Cas protein and second Cas protein are the same or different, wherein the nucleobase deaminase inhibitor domain is connected to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein optionally by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof, wherein the mgRNA further comprises a second protein-binding motif and a third protein-binding motif, wherein the second RNA binding domain binds to the second protein-binding motif, wherein the third RNA binding domain binds to the third protein-binding motif, and wherein the second and third RNA binding domains are the same or different, and the second and third protein-binding motifs are the same or different.

A “protease” refers to an enzyme that catalyzes proteolysis. A “cleavage site for a protease” refers to a short peptide that the protease recognizes, and within the short peptide creates a proteolytic cleavage. Non-limiting examples of proteases include TEV protease, TuMV protease, PPV protease, PVY protease, ZIKV protease, and WNV protease. The protein sequences of example proteases and their corresponding cleavage sites are provided in Table 7.

Table 7 Exemplary proteases and their cleavage sites

A “nucleobase deaminase inhibitor” or an “inhibitory domain” refers to a protein or a protein domain that inhibits the deaminase activity of a nucleobase deaminase.

Table 8 shows 44 proteins/domains that have significant sequence homology to mA3-CDA2 core sequence and Table 9 shows 43 proteins/domains that have significant sequence homology to hA3B-CDA1. All of these proteins and domains, as well as their variants and equivalents, are contemplated to have nucleobase deaminase inhibition activities.

Table 8

Table 9

The term "nucleobase deaminase" as used herein, refers to a group of enzymes that catalyze the hydrolytic deamination of nucleobases such as cytidine, deoxycytidine, adenosine and deoxyadenosine. Non-limiting examples of nucleobase deaminases include cytidine deaminases and adenosine deaminases.

Some of the nucleobase deaminases have a single, catalytic domain, while others also have other domains, such as an inhibitory domain as described in WO2020156575A1. In some embodiments, therefore, the gene editing system disclosed herein only includes the catalytic domain, such as mouse A3 cytidine deaminase domain 1 (mA3-CDA1, SEQ ID NO: 224) and human A3B cytidine deaminase domain 2 (hA3B-CDA2, SEQ ID NO: 225) . In some embodiments, the gene editing system disclosed herein includes at least a catalytic core of the catalytic domain. For instance, when mA3-CDA1 was truncated at residues 196/197 the CDA1 domain still retained substantial editing efficiencies.

Table 10

“Cytidine deaminase” refers to enzymes that catalyze the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Cytidine deaminases maintain the cellular pyrimidine pool. A family of cytidine deaminases is APOBEC ( “apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like” ) . Members of this family are C-to-U editing enzymes. Some APOBEC family members have two domains, one domain of APOBEC like proteins is the catalytic domain, while the other domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination. RNA editing by APOBEC-1 requires homodimerisation and this complex interacts with RNA binding proteins to form the editosome.

Non-limiting examples of APOBEC proteins include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase (AID) .

Various mutants of the APOBEC proteins are also known that have brought about different editing characteristics for base editors. For instance, for human APOBEC3A, certain mutants (e.g., W98Y, Y130F, Y132D, W104A, D131Y and P134Y) even outperform the wildtype human APOBEC3A in terms of editing efficiency or editing window. Accordingly, the term APOBEC and each of its family member also encompasses variants and mutants that have certain level (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%) of sequence identity to the corresponding wildtype APOBEC protein or the catalytic domain and retain the cytidine deaminating activity. The variants and mutants can be derived with amino acid additions, deletions and/or substitutions. Such substitutions, in some embodiments, are conservative substitutions.

In some embodiments, the cytidine deaminase comprises an amino acid sequence of any one of SEQ ID NOs: 477-507, 789-792. (Table 15)

Table 15

In some embodiments, the nucleotide deaminase is an adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an amino acid sequence of any one of SEQ ID NOs: 384-476. (Table 16)

Table 16

The “Uracil Glycosylase Inhibitor” (UGI) , which can be prepared from Bacillus subtilis bacteriophage PBS1, is a small protein (9.5 kDa) which inhibits E. coli uracil-DNA glycosylase (UDG) as well as UDG from other species. Inhibition of UDG occurs by reversible protein binding with a 1 : 1 UDG : UGI stoichiometry. UGI is capable of dissociating UDG-DNA complexes. A non-limiting example of UGI is found in Bacillus phage AR9 (YP_009283008.1) . In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NO: 226 or has at least 70%, 75%, 80%, 85%, 90%or 95%sequence identity to SEQ ID NO: 226 and retains the uracil glycosylase inhibition activity.

A “nuclear localization signal or sequence” (NLS) is an amino acid sequence that tags a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. A non-limiting example of NLS is the internal SV40 nuclear localization sequence (iNLS) .

The term “Cas protein” or “clustered regularly interspaced short palindromic repeats (CRISPR) -associated (Cas) protein” refers to RNA-guided DNA endonuclease enzymes associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, as well as other bacteria. Cas proteins include Cas9 proteins, Cas12a (Cpf1) proteins, Cas12b (formerly known as C2c1) proteins, Cas13 proteins and various engineered counterparts. Example Cas proteins include SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpf1, FnCpf1, VQR SpCas9, EQR SpCas9, VRER SpCas9, SpCas9-NG, xSpCas9, RHA FnCas9, KKH SaCas9, NmeCas9, StCas9, CjCas9, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b, LsCas12b, RfCas13d, LwaCas13a, PspCas13b, PguCas13b, RanCas13b and those provided in Table 11 below.

Table 11 Exemplary Cas Proteins

In some embodiments, the Cas protein is a Cas9, a dead Cas9 (dCas9) , or a Cas9 nickase (nCas9) .

In some embodiments, the Cas protein is a nCas9. In some embodiments, the nCas9 protein is a nCas9-D10A protein. In some embodiments, the nCas9-D10A protein has an amino acid sequence of SEQ ID NO: 227.

In some embodiments, the Cas protein comprises an amino acid sequence of any one of SEQ ID NOs: 333-383 (Table 14)

Table 14

In some embodiments, the first protein-binding RNA motif and the first RNA binding domain, the second protein-binding RNA motif and the second RNA binding domain, and the third protein-binding RNA motif and the third RNA binding domain, are each independently selected from the group consisting of a MS2 phage operator stem-loop and MS2 coat protein (MCP) or an RNA-binding section thereof; a BoxB and N22P or an RNA-binding section thereof; a telomerase Ku binding motif and Ku protein or an RNA-binding section thereof; a telomerase Sm7 binding motif and Sm7 protein or an RNA-binding section thereof; a PP7 phage operator stem-loop and PP7 coat protein (PCP) or an RNA-binding section thereof; a SfMu phage Com stem-loop and Com RNA binding protein or an RNA-binding section thereof; and a non-natural RNA aptamer and corresponding aptamer ligand or an RNA-binding section thereof. See Table 12.

Table 12

For any protein of the present disclosure, biological equivalents thereof are also provided. In some embodiments, the biological equivalents have at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%sequence identity with the reference protein. Preferably, the biological equivalents retain the desired activity of the reference protein. In some embodiments, the biological equivalents are derived by including one, two, three, four, five, or more amino acid additions, deletions, substitutions, or the combinations thereof. In some embodiments, the substitution is a conservative amino acid substitution.

In some embodiments of the gene editing systems described herein, the guide RNA (the (main) single guide RNA and/or the helper guide RNA) is a dual-RNA structure formed by a ligand-bound CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) . In some embodiments, the crRNA comprises a spacer sequence and is capable of forming a base-pair structure with the tracrRNA, and wherein the base-pair structure binds to a Cas protein. In some embodiments, the crRNA further comprises a linker sequence which comprises a protein-binding motif. For the purpose of the present disclosure, when the guide RNA is a dual-RNA structure of crRNA and tracrRNA, the “CRISPR motif” refers to the base-pair structure formed between the crRNA and the tracrRNA.

In some embodiments, the gene editing system is a LIGO-RNA-based gene editing system, as described in PCT/CN2023/096482, which is incorporated herein by reference in its entirety. A person skilled in the art would be able to design the corresponding crRNA-tracrRNA pair based on the sgRNA and hsgRNA disclosed herein.

In the LigoRNA-based gene editing system, at least one guide RNA is a LigoRNA. A LigoRNA system comprises a dual-RNA structure, which can be used as a guide RNA in CRISPR-based gene editing systems. The dual-RNA structure can be formed by a ligand-bound CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) . For example, the LigoRNA system comprises an hgRNA set of a hcrRNA and a tracrRNA, and an mgRNA set of mcrRNA and a tracrRNA. Preferably, all of these RNA molecules are not longer than 100 nucleotides.

Since the LigoRNA system is formed by two short RNAs, it helps to solve the problem of synthesizing long single guide RNAs in previous gene editing systems. Chemically synthesized RNAs over 100 nt demonstrated much lower yield and purity, resulting in challenges for large-scale production and cost control.

Original types of crRNA and tracrRNA are capable of guiding nCas9-mediated DNA location. The crRNAs and the tracrRNAs in the LigoRNA system are further modified. In some embodiments, an MS2 or boxB hairpin is fused to crRNA in multiple different sites. In some embodiments, at least one nucleotide in the crRNAs and the tracrRNAs is modified, such as by a 2’-O-methyl modification and/or 3’-phosphorothioate modification.

In some embodiments, the crRNA comprises a spacer sequence and a linker sequence, wherein the linker sequence comprises at least one protein-binding motif, wherein the protein-binding motif is an RNA aptamer motif. In some embodiments, the protein binding motif is selected from MS2, PP7, boxB, SfMu hairpin motif, telomerase Ku, and Sm7 binding motif, or a variant thereof. Aptamers are single-stranded oligonucleotides that fold into defined architectures and selectively bind to a specific target, including proteins, peptides, carbohydrates, small molecules, toxins, and even live cells.

In some embodiments, the crRNA is capable of forming a base-pair structure with a trans-activating crRNA (tracrRNA) . In some embodiments, the tracrRNA has an sequence of SEQ ID NO: 655.

In some embodiments, the crRNA comprises at least one nucleotide with modification. In some embodiments, the modification is selected from 2’-O-alkyl, 2’-substituted alkoxy, 2’-substituted alkyl, 2’-halo, 3’-phosphorothioate, bridged nucleic acid (BNA) , and locked nucleic acid (LNA) . In some embodiments, the at least one nucleotide with modification is any one of the first three nucleotides from 3’-end of the engineered crRNA.

In some embodiments, the tracrRNA comprises at least one nucleotide with modification. In some embodiments, the modification is selected from 2’-O-alkyl, 2’-substituted alkoxy, 2’-substituted alkyl, 2’-halo, 3’-phosphorothioate, bridged nucleic acid (BNA) , and locked nucleic acid (LNA) . In some embodiments, the at least one nucleotide with modification is any one of the first three nucleotides from 3’-end of the engineered tracrRNA.

In some embodiments, the crRNA and/or tracrRNA comprises at least one nucleotide with modification. In some embodiments, the modification is selected from 2’-O-alkyl (such as 2’-O-methyl) , 2’-substituted alkoxy, 2’-substituted alkyl, 2’-halo (such as 2’-fluoro) , 3’-phosphorothioate, bridged nucleic acid (BNA) , and locked nucleic acid (LNA) . In some embodiments, the crRNA and/or tracrRNA comprises nucleotides comprising 2’-O-methyl and 3’-phosphorothioate. In some embodiments, the first three nucleotides from the 5’-end of the crRNA and/or tracrRNA are modified with 2’-O-methyl and 3’-phosphorothioate. In some embodiments, the first three nucleotides from the 3’-end of the crRNA and/or tracrRNA are modified with 2’-O-methyl, and the second to fourth nucleotides from the 3’-end of the crRNA and/or tracrRNA are modified with 3’-phosphorothioate. In some embodiments, the first three nucleotides from the 5’-end of the crRNA and/or tracrRNA are modified with 2’-O-methyl and 3’-phosphorothioate, and the first three nucleotides from the 3’-end of the crRNA and/or tracrRNA are modified with 2’-O-methyl, and the second to fourth nucleotides from the 3’-end of the crRNA and/or tracrRNA are modified with 3’-phosphorothioate.

In some embodiments, is the present disclosure provides a tBE system comprising two LigoRNA structures: an mcrRNA-tracrRNA base-paired structure and an hcrRNA-tracrRNA base-paired structure. In some embodiments, the mcrRNA contains a boxB hairpin to generate an R-loop region for intended base editing and the hcrRNA contains an MS2 hairpin to recruit a nucleotide deaminase (e.g., an APOBEC linked to a nucleobase deaminase inhibitor (e.g., a cytosine deaminase inhibitor (dCDI) ) domain through a cleavage site such as a TEV protease cleavage site. For example, to cleave off the dCDI domain at the on-target sites, an N22p-fused TEVc is recruited by the boxB-containing mcrRNA, working as the key in tBE system with free TEVn. In some embodiments, mcrRNA and hcrRNA form a base-paired structure with the same tracrRNA to locate a target DNA, and the dCDI domain is cleaved off at the target site to induce efficient base editing.

In some embodiments of the gene editing system described herein, the gene editing system comprises

a. an hcrRNA comprising a first spacer sequence and a first linker sequence, wherein the first linker sequence comprises a first protein-binding motif,

b. an mcrRNA comprising a second spacer sequence and a second linker sequence, wherein the second linker sequence comprises a second protein-binding motif,

c. a first tracrRNA which is capable of forming a first base-pair structure with the hcrRNA,

d. a second tracrRNA which is capable of forming a second base-pair structure with the mcrRNA,

e. a first CRISPR-associated protein (Cas protein) , or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first base-pair structure,

f. a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second base pair structure,

g. a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif, wherein the first Cas protein and the second Cas protein are the same or different, and the first tracrRNA and the second tracrRNA are the same or different.

In some embodiments of the gene editing system described herein, the gene editing system further comprises

a. a protease, or a polynucleotide encoding the protease, and

b. a nucleobase deaminase inhibitor domain,

wherein the nucleobase deaminase inhibitor domain is connected to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein optionally by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof.

g. a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif,

h. a protease, or a polynucleotide encoding the protease,

i. a nucleobase deaminase inhibitor domain, and

j. a second fusion protein comprising the protease and a second RNA binding domain, or a polynucleotide encoding the second fusion protein,

wherein the first Cas protein and the second Cas protein are the same or different, and the first tracrRNA and the second tracrRNA are the same or different,

wherein the nucleobase deaminase inhibitor domain is connected to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein optionally by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof,

wherein the protease and the second RNA binding domain are optionally connected by a linker, and

wherein the second RNA binding domain binds to the second protein-binding motif.

In some embodiments of the gene editing system described herein, the protease is split into a first protease fragment and a second protease fragment, wherein the first and/or second protease fragment alone is not able to cleave the cleavage site.

In some embodiments of the gene editing system described herein, wherein the gene editing system comprises

h. a protease, or a polynucleotide encoding the protease, wherein the protease is split into a first protease fragment and a second protease fragment, wherein the first and/or second protease fragment alone is not able to cleave the cleavage site,

i. a nucleobase deaminase inhibitor domain,

j. a second fusion protein comprising the first protease fragment and a second RNA binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA binding domain are optionally connected by a linker, and

k. a third fusion protein comprising the second protease fragment and a third RNA binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA binding domain are optionally connected by a linker,

wherein the mcrRNA further comprises a third protein-binding motif,

wherein the second RNA binding domain binds to the second protein-binding motif, and

wherein the third RNA binding domain binds to the third protein-binding motif.

i. a nucleobase deaminase inhibitor domain,

wherein the mcrRNA further comprises a third protein-binding motif,

wherein the second RNA binding domain binds to the second protein-binding motif,

wherein the third RNA binding domain binds to the third protein-binding motif, and

wherein the second and the third RNA binding domains are the same or different, and the second and the third protein-binding motifs are the same or different.

i. a nucleobase deaminase inhibitor domain,

j. a second fusion protein comprising the first protease fragment and a second RNA binding domain, or a polynucleotide encoding the second fusion protein,

wherein the first protease fragment and the second RNA binding domain are optionally connected by a linker, and

In some embodiments, the mcrRNA and the hcrRNA are

SEQ ID NO. 639 and SEQ ID NO: 647, respectively; or

SEQ ID NO. 640 and SEQ ID NO: 648, respectively; or

SEQ ID NO. 641 and SEQ ID NO: 649, respectively; or

SEQ ID NO. 642 and SEQ ID NO: 650, respectively; or

SEQ ID NO. 643 and SEQ ID NO: 651, respectively; or

SEQ ID NO. 644 and SEQ ID NO: 652, respectively; or

SEQ ID NO. 645 and SEQ ID NO: 653, respectively; or

SEQ ID NO. 646 and SEQ ID NO: 654, respectively.

In some embodiments, the tracrRNA is SEQ ID NO: 655.

Polynucleotides

The polynucleotides disclosed herein can be obtained by methods known in the art. For example, the polynucleotide can be obtained from cloned DNA (e.g., from a DNA library) , by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA or fragments thereof, purified from the desired cell. When the polynucleotides are produced by recombinant means, any method known to those skilled in the art for identification of nucleic acids that encode desired genes can be used. Any method available in the art can be used to obtain a full length (i.e., encompassing the entire coding region) cDNA or genomic DNA encoding a desired protein, such as from a cell or tissue source. Modified or variant polynucleotides can be engineered from a wildtype polynucleotide using standard recombinant DNA methods. Polynucleotides can be cloned or isolated using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening, antibody-based screening, and activity-based screening.

Methods for amplification of polynucleotides can be used to isolate polynucleotides encoding a desired protein, including for example, polymerase chain reaction (PCR) methods. PCR can be carried out using any known methods or procedures in the art. Exemplary methods include use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp) . A nucleic acid containing gene of interest can be used as a source material from which a desired polypeptide-encoding nucleic acid molecule can be amplified. For example, DNA and mRNA preparations, cell extracts, tissue extracts from an appropriate source (e.g., testis, prostate, breast) , fluid samples (e.g., blood, serum, saliva) , samples from healthy and/or diseased subjects can be used in amplification methods. The source can be from any eukaryotic species including, but not limited to, vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, and other primate sources. Nucleic acid libraries also can be used as a source material. Primers can be designed to amplify a desired polynucleotide. For example, primers can be designed based on expressed sequences from which a desired polynucleotide is generated. Primers can be designed based on back-translation of a polypeptide amino acid sequence. If desired, degenerate primers can be used for amplification. Oligonucleotide primers that hybridize to sequences at the 3’ and 5’ termini of the desired sequence can be uses as primers to amplify by PCR from a nucleic acid sample. Primers can be used to amplify the entire full-length polynucleotide, or a truncated sequence thereof. Nucleic acid molecules generated by amplification can be sequenced and confirmed to encode a desired polypeptide.

Vectors

In some embodiments, the vector is a plasmid or a viral vector.

In some embodiments, the vector is a polycistronic vector.

Any methods known in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors comprising a polynucleotide disclosed herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo (genetic) recombination. The polynucleotide disclosed herein can be operably linked to control sequences in the expression vector (s) to ensure protein expression. Such control sequences may include, but are not limited to, leader or signal sequences, promoters (e.g., naturally associated or heterologous promoters) , ribosomal binding sites, enhancer or activator elements, translational start and termination sequences, and transcription start and termination sequences, and are chosen to be compatible with the host cell chosen to express the proteins. Constitutive or inducible promoters as known in the art are also contemplated. The promoters may be either naturally occurring promoters, hybrid promoters that combine elements of more than one promoter, or synthetic promoters. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome such as in a gene locus. In some embodiment, the expression vector includes a selectable marker gene to allow the selection of transformed host cells. In some embodiments, the vector is an expression vector comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory control sequence. Regulatory control sequences for use herein include promoters, enhancers, and other expression control elements. In some embodiments, the expression vector is designed for the choice of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the vector's copy number, the ability to control that copy number, and/or the expression of any other protein encoded by the vector, such as antibiotic markers.

The vector can include, but is not limited to, viral vectors and plasmid DNA. Viral vectors can include, but are not limited to, adenoviral vectors, lentiviral vectors, retroviral vectors, and adeno-associated viral vectors. Commonly, expression vectors contain selection markers such as ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences. Suitable vectors, promoter, and enhancer elements are known in the art; many are commercially available for generating subject recombinant constructs. In some embodiments, the vector is a polycistronic vector. In some embodiments, the vector is a bicistronic vector or a tricistronic vector. Bicistronic or polycistronic expression vectors may include (1) multiple promoters fused to each of the open reading frames; (2) insertion of splicing signals between genes; (3) fusion of genes whose expressions are driven by a single promoter; and (4) insertion of proteolytic cleavage sites between genes (self-cleavage peptide) or insertion of internal ribosomal entry sites (IRESs) between genes.

A polycistronic vector is used to co-express multiple genes in the same cell. Two strategies are most commonly used to construct a multicistronic vector. First, an Internal Ribosome Entry Site (IRES) element is typically used for bi-cistronic vectors. The IRES element, acting as another ribosome recruitment site, allows initiation of translation from an internal region of the mRNA. Thus, two proteins are translated from one mRNA. IRES elements are quite large (usually 500-600 bp) (Pelletier et al., 1988; Jang et al., 1988) . The engineered CD47 proteins disclosed herein have a smaller size compared to the wild-type full-length human CD47, and thus could be used with IRES element in a multicistronic vectors having limited packaging capacity.

Cells

In another aspect, the present disclosure provides a cell comprising one or more of the gene editing systems disclosed herein.

In another aspect, the present disclosure provides a cell comprising the kit disclosed herein.

In some embodiments, the cell is infected by an HBV. A cell is infected by an HBV when an HBV virion enters the cell.

In some embodiments, the cell comprises an HBV integrated DNA.

In some embodiments, the cell is a liver cell. In some embodiments, the cell is a hepatocyte. As the largest solid organ in the body, the liver consists of multiple cell types that are responsible for the organism-level functions of metabolism, detoxification, coagulation, and immune response. Four major liver cell types in liver are hepatocytes (HCs) , hepatic stellate cells (HSCs) , Kupffer cells (KCs) , and liver sinusoidal endothelial cells (LSECs) . They spatiotemporally cooperate to shape and maintain liver functions. Hepatocytes constitute about 70%of the total liver cell population. As the parenchymal portion of the liver, hepatocytes are primarily engaged in the basic functions of the liver, including lipid metabolism, drug metabolism, and the secretion of coagulation and complement factors. Kupffer cells, which represent one-third of the nonparenchymal cells in the liver, serve as immune sentinels. Although hepatic stellate cells comprise only 5%of the liver cells, they play central roles in vitamin A and lipid storage. Liver sinusoidal endothelial cells, which comprise the largest part (50%) of liver nonparenchymal cells, separate the underlying hepatocytes from the sinusoidal lumen.

Compositions

As used herein, the term “composition” includes, but is not limited to, a pharmaceutical composition. A “pharmaceutical composition” refers to an active pharmaceutical agent formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the disclosure may be administered in combination with other agents, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy. The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The compositions may also comprise a pharmaceutically acceptable carrier, diluent, or excipient. As used herein “pharmaceutically acceptable carrier, diluent, or excipient” includes, without limitation, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter; waxes; animal and vegetable fats; paraffins; silicones; bentonites; silicic acid; zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate, and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations.

The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline; Ringers solution; isotonic sodium chloride; fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium; polyethylene glycols; glycerin; propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

The composition may be suitably developed for intravenous, intratumoral, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration.

Methods of Treatment

In another aspect, the present disclosure provides a method for disrupting an HBV gene in a cell, comprising introducing into the cell one or more of the gene editing systems disclosed herein. In some embodiments, the present disclosure provides a method for disrupting an HBV S gene comprising introducing into the cell the gene editing system disclosed herein, wherein the combination of mgRNA and hgRNA are selected from Table 2 or Table 3. In some embodiments, the present disclosure provides a method for disrupting an HBV P gene comprising introducing into the cell the gene editing system disclosed herein, wherein the combination of mgRNA and hgRNA are selected from Table 4. In some embodiments, the present disclosure provides a method for disrupting an HBV X gene comprising introducing into the cell the gene editing system disclosed herein, wherein the combination of mgRNA and hgRNA are selected from Table 5. In some embodiments, the present disclosure provides a method for disrupting an HBV C gene comprising introducing into the cell the gene editing system disclosed herein, wherein the combination of mgRNA and hgRNA are selected from Table 6.

In another aspect, the present disclosure provides a method for treating HBV infection in a subject, comprising disrupting an HBV gene in a cell of the subject with any one of the methods disclosed herein.

In another aspect, the present disclosure provides a method for treating or preventing chronic hepatitis B (CHB) , liver cirrhosis, hepatocellular carcinoma, and/or liver failure. Hepatitis B is a serious liver infection caused by HBV. For most people, hepatitis B is short term (acute) and lasts less than six months. But for others, the infection becomes chronic, meaning it lasts more than six months. about 5%to10%of adults and up to 90%of young children fail to produce an immune response adequate to clear the virus, and thus subsequently develop chronic hepatitis B (CHB) , which often progresses to liver cirrhosis, hepatocellular carcinoma, and liver failure. Liver cirrhosis refers to the impaired liver function caused by the formation of scar tissue known as fibrosis due to damage caused by liver disease. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Hepatocellular carcinoma occurs most often in people with chronic liver diseases, such as cirrhosis caused by HBV infection.

Table 13

EXAMPLE

To apply the tBE system to generate stop codons in HBV gene, 166 pairs of mgRNA/hgRNAs that target HBV genes were designed, with 83 pairs comprising 20-nt hgRNAs and 83 pairs comprising 10-nt hgRNAs. The 83 pairs of mgRNA/hgRNAs comprising 20-nt hgRNAs were tested and demonstrated to have good editing efficiency. The mgRNA/hgRNA pairs comprising 10-nt hgRNAs are contemplated to have substantially the same editing efficiency with their respective 20-nt counterparts. Gene editing results using several exemplary pairs of mgRNA/hgRNAs are shown in Figs. 2-9 and 11-17. Specifically, tBE systems were used to induce C-to-T base editing in the codons of CAA (Gln) , CAG (Gln) , or CGA (Arg) in HBV genes to create a TAA, TAG, or TGA stop codon (Figs. 2-9, 11-13, 14A, 15A, 16A, and 17A) . tBE systems were also used to induce G-to-A (C-to-T on the opposite strand) base editing in the codons of TGG (Trp) in HBV genes to create a TAA, TAG, or TGA stop codon (Figs. 2-9, 11-13, 14A, 15A, 16A, and 17A) . To apply the tBE system to generate stop codons in HBV gene of six different distinct HBV genotypes (Ato F) , several pairs of mgRNA/hgRNAs that target HBV genes were designed. Gene editing results using several exemplary pairs of mgRNA/hgRNAs are shown in Figs. 12. To apply the LigoRNA based-tBE system to generate stop codons in HBV gene, 8 pairs of mcrRNA/hcrRNAs with tracrRNA that target HBV genes were designed. Gene editing results using several exemplary pairs of mcrRNA/hcrRNAs with tracrRNA are shown in Figs. 13. Relative hepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg) loss were detected after transfection (Figs. 14B, 15B, 16B, and 17B) . Multiplexing two pairs of mgRNA/hgRNAs introducing stop codons in HBV genes S region and C region with tBE leads to a simultaneous reduction of HBsAg and HBeAg (Figs. 18 and 19) .

To apply the tBE system to generate stop codons in HBV NTCP receptor, 44 pairs of mgRNA/hgRNAs that target HBV genes were designed, with 22 pairs comprising 20-nt hgRNAs and 22 pairs comprising 10-nt hgRNAs. The 22 pairs of mgRNA/hgRNAs comprising 20-nt hgRNAs were tested and demonstrated to have good editing efficiency. The mgRNA/hgRNA pairs comprising 10-nt hgRNAs are contemplated to have substantially the same editing efficiency with their respective 20-nt counterparts. Gene editing results using several exemplary pairs of mgRNA/hgRNAs are shown in Figs. 20 and 21. Specifically, tBE systems were used to induce C-to-T base editing in the codons of CAA (Gln) , CAG (Gln) , or CGA (Arg) in HBV genes to create a TAA, TAG, or TGA stop codon (Figs. 20 and 21) . tBE systems were also used to induce G-to-A (C-to-T on the opposite strand) base editing in the codons of TGG (Trp) in HBV genes to create a TAA, TAG, or TGA stop codon (Figs. 20, 21) .

HEK293FT or HepG2 cell lines that stably express P gene, S gene, X gene, and C gene of HBV (HBV-P, HBV-S, HBV-X and HBV-C) , respectively, were established for assessing the efficiency of tBE at the potential target sites.

Genomic DNA was extracted 72 hours after transfecting plasmids into cells, and the C-to-T editing efficiencies of tBE systems comprising different hgRNAs with one mgRNA at target sites were compared. From the sanger sequencing results, it was found that the tBE systems provided high base editing efficiencies at these target sites, resulting in the generation of stop codons in HBV genes.

Plasmid construction

Primer sets (hsg-HBV-S-CAG-1-U1_FOR/sg1-HBV-S-CAG-1_REV) were used to amplify the fragment hsg-sg1-U1-HBV-S-MS2 (the operator in hgRNA scaffold) -U6 (mgRNA promoter) -sg1-HBV-S-CAG-1 using the template pUC57-mgRNA-MS2-U6. The fragment hsg-sg1-U1-HBV-S-MS2-U6-sg1-HBV-S-CAG-1 was then ligated into BsmBI-linearized U6-ccdB-boxB-tBE-V5 to generate the vector ptBE-V5-HBV-S-CAG-1-U1. Other combinations with different on-target hgRNA and mgRNA were constructed using the same strategy, respectively.

Cell culture and transfection

293FT cells or HepG2 cells were maintained in DMEM + 10%FBS and regularly tested to exclude mycoplasma contamination. For establishing 293FT or HepG2 cell lines stably expressing HBV-S, HBV-P, HBV-X or HBV-C, 5× 10⁵ 293FTor HepG2 cells per well were seeded in a 6-well plate and transfected with 50ul serum-free Opti-MEM containing 5 μl LIPOFECTAMINE LTX, 2 μl LIPOFECTAMINE plus and 2 μg HBV-S, HBV-P, HBV-X or HBV-C plasmid, respectively. After 24h, cells were selected with G418 or Blasticidin for 2 weeks to establish 293FT-HBV-S, 293FT-HBV-P, 293FT-HBV-X and 293FT-HBV-C stable cell lines or HepG2-HBV-C-S, HepG2-HBV-C-C and HepG2-HBV-D-S, HepG2-HBV-D-C stable cell lines. The integration of HBV genes was finally quantified by qRT-PCR to make sure the stable cell lines were successfully established. For base editing with transformer BEs, the stable cells were seeded in a 24-well plate at a density of 1 × 10⁵ per well and transfected with 250 μl serum-free Opti-MEM containing 2.5 μl LIPOFECTAMINE LTX, 1 μl LIPOFECTAMINE plus, 0.5 μg tBE-V5 expression vector, 0.5 μg pEFS-nSpCas9, pEFS-nSpCas9-NG or nSpCas9-SpG expression vector. After 24 h, puromycin was added to the medium at a final concentration of 4 μg/ml. After another 48 h, the genomic DNA was extracted from the cells using QuickExtractT DNA Extraction Solution for subsequent sequencing analysis. Target genomic sequences were PCR-amplified using high-fidelity DNA polymerase PrimeSTAR HS with primer sets flanking the examined mgRNA target sites.

For electroporation, chemically modified sgRNA (2’-O-methyl and 3’ phosphorothioate modifications in the first and last three nucleotides) was synthesized. HepG2, HepG2.2.15 or PLC cell were electroporated with the end-modified guide RNA and the mRNAs described above. Electroporation was performed using Lonza 4D Nucleofector by using officially recommended program (e.g., EH-100) . For 20-μl Nucleocuvette Strips, 0.2 million cells were resuspended in 20 μl SF Cell Line 4D-Nucleofector buffer and about RNA complex were added. The editing frequencies of target sequence were measured with cells cultured in medium 96 hours after electroporation.

HBsAg and HBeAg analysis

HBsAg of HBV-C or D genotype stably transfected HepG2 were measured in whole cell protein lysis. HBeAg of HBV-C or D genotype stably transfected HepG2 were measured in cell culture supernatant. HBsAg and HbeAg of HepG2.2.15 were measured in cell culture supernatant. They were test by ELISA kit according to the manufacturer’s protocol. The luminescence signal was collected by spectraMax M5e microplate reader.

Base substitution frequency at each target sites was calculated by EditR analysis. See http: //baseeditr. com/.

Base substitution calculation, statistics analysis, and other relevant steps for obtaining the data as illustrated in Figs. 2-9 are essentially the same as disclosed in the “Methods” section of Wang, Lijie, et al., Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations, Nature Cell Biology 23.5 (2021) : 552-563, the content of which is incorporated herein by reference in its entirety.

Gene editing results obtained from the above experiments are illustrated in Figs. 2-9.

While the disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

Claims

A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting a human hepatitis B virus (HBV) S gene and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 1-8.
The gene editing system of claim 1, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting a PreS1 region on an HBV S gene and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 9-12.
The gene editing system of claim 3, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV P gene on and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 13-30.
The gene editing system of claim 5, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV X gene and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 31-32.
The gene editing system of claim 7, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 33-38.
The gene editing system of claim 9, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-A genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 515-518.
The gene editing system of claim 11, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-A genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 519.
The gene editing system of claim 13, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-A genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 520-522.
The gene editing system of claim 15, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-B genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 531-534.
The gene editing system of claim 17, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-B genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 535.
The gene editing system of claim 19, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-B genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 536-538.
The gene editing system of claim 21, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-C genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 547-550.
The gene editing system of claim 23, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-C genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NOs: 551.
The gene editing system of claim 25, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-C genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 552-554.
The gene editing system of claim 27, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 563-566.
The gene editing system of claim 29, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 567.
The gene editing system of claim 31, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 568-570.
The gene editing system of claim 33, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-E genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 579-582.
The gene editing system of claim 35, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-E genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NOs: 583.
The gene editing system of claim 37, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-E genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 584-586.
The gene editing system of claim 39, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-F genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 595-598.
The gene editing system of claim 41, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-F genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence of SEQ ID NO: 599.
The gene editing system of claim 43, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-F genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 600-602.
The gene editing system of claim 45, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-C genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence selected from SEQ ID NOs: 611-613.
The gene editing system of claim 47, wherein the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-C genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence of SEQ ID NO: 614.
The gene editing system of claim 49, wherein the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-C genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence selected from SEQ ID NOs: 615-617.
The gene editing system of claim 51, wherein the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV S gene of HBV-D genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence selected from SEQ ID NOs: 625-627.
The gene editing system of claim 53, wherein the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV Pre-S1 gene of HBV-D genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence of SEQ ID NO: 628.
The gene editing system of claim 55, wherein the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV C gene of HBV-D genotype, wherein the nucleic acid sequence of the mgRNA comprises a sequence selected from SEQ ID NOs: 629-631.
The gene editing system of claim 57, wherein the nucleic acid sequences of the mgRNA and the hgRNA comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a main crRNA (mcrRNA) and a first tracrRNA, wherein the hgRNA comprises a helper crRNA (hcrRNA) and a second tracrRNA, wherein the mgRNA targets an HBV S gene of HBV-D genotype, wherein the nucleic acid sequence of the mcrRNA comprises a sequence selected from SEQ ID NOs: 639-642.
The gene editing system of claim 59, wherein the nucleic acid sequences of the mcrRNA and the hcrRNA comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a main crRNA (mcrRNA) and a first tracrRNA, wherein the hgRNA comprises a helper crRNA (hcrRNA) and a second tracrRNA, wherein the mgRNA targets an HBV Pre-S1 gene of HBV-D genotype, wherein the nucleic acid sequence of the mcrRNA comprises a sequence of SEQ ID NO: 643.
The gene editing system of claim 61, wherein the nucleic acid sequences of the mcrRNA and the hcrRNA comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a main crRNA (mcrRNA) and a first tracrRNA, wherein the hgRNA comprises a helper crRNA (hcrRNA) and a second tracrRNA, wherein the mgRNA targets an HBV C gene of HBV-D genotype, wherein the nucleic acid sequence of the mcrRNA comprises a sequence selected from SEQ ID NOs: 644-646.
The gene editing system of claim 63, wherein the nucleic acid sequences of the mcrRNA and the hcrRNA comprise respectively:
The gene editing system of any one of claims 59-64, wherein at least one of the first and second tracrRNA comprises a sequence of SEQ ID NO: 655.
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 656-664.
The gene editing system of claim 66, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting an HBV gene of HBV-D genotype, and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 689-692.
The gene editing system of claim 68, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
The gene editing system of any one of claims 66-69, wherein the gene editing system induces missense mutation.
A gene editing system comprising a main guide RNA (mgRNA) and a helper guide RNA (hgRNA) , or at least one DNA polynucleotide encoding the mgRNA and/or the hgRNA, wherein the mgRNA comprises a mgRNA spacer targeting a NTCP receptor and the hgRNA comprises a hgRNA spacer, wherein the nucleic acid sequence of the mgRNA spacer comprises a sequence selected from SEQ ID NOs: 700-714.
The gene editing system of claim 71, wherein the nucleic acid sequences of the mgRNA spacer and the hgRNA spacer comprise respectively:
The gene editing system of any one of claims 1-72, comprising

a. the hgRNA comprising a first CRISPR motif, the hgRNA spacer, and a first protein-binding motif, or a DNA polynucleotide encoding the hgRNA,

b. the mgRNA comprising a second CRISPR motif and the mgRNA spacer, or a DNA polynucleotide encoding the mgRNA,

c. a first CRISPR-associated protein (Cas protein) , or a polynucleotide encoding the first Cas protein, wherein the first Cas protein binds to the first CRISPR motif,

d. a second Cas protein, or a polynucleotide encoding the second Cas protein, wherein the second Cas protein binds to the second CRISPR motif,

e. a first fusion protein comprising a nucleobase deaminase or a catalytic domain thereof and a first RNA binding domain, or a polynucleotide encoding the first fusion protein, wherein the nucleobase deaminase or the catalytic domain thereof and the first RNA binding domain are optionally connected by a linker, and wherein the first RNA binding domain binds to the first protein-binding motif.

wherein the first Cas protein and second Cas protein are the same or different.
The gene editing system of claim 73, further comprising

a. a protease, or a polynucleotide encoding the protease, and

b. a nucleobase deaminase inhibitor domain,

wherein the nucleobase deaminase inhibitor domain is connected to the nucleobase deaminase or the catalytic domain thereof in the first fusion protein optionally by a linker, and wherein there is a cleavage site for the protease between the nucleobase deaminase inhibitor domain and the nucleobase deaminase or the catalytic domain thereof.
The gene editing system of claim 74, further comprising

a second fusion protein comprising the protease and a second RNA binding domain, or a polynucleotide encoding the second fusion protein,

wherein the protease and the second RNA binding domain are optionally connected by a linker,

wherein the mgRNA further comprises a second protein-binding motif,

and wherein the second RNA binding domain binds to the second protein-binding motif.
The gene editing system of claim 74, wherein the protease is split into a first protease fragment and a second protease fragment, wherein the first and/or second protease fragment alone is not able to cleave the cleavage site.
The gene editing system of claim 76, further comprising

a. a second fusion protein comprising the first protease fragment and a second RNA binding domain, or a polynucleotide encoding the second fusion protein, wherein the first protease fragment and the second RNA binding domain are optionally connected by a linker, and

b. a third fusion protein comprising the second protease fragment and a third RNA binding domain, or a polynucleotide encoding the third fusion protein, wherein the second protease fragment and the third RNA binding domain are optionally connected by a linker,

wherein the mgRNA further comprises a second protein-binding motif and a third protein-binding motif,

wherein the second RNA binding domain binds to the second protein-binding motif, and

wherein the third RNA binding domain binds to the third protein-binding motif.
The gene editing system of claim 77, wherein the second and third RNA binding domains are the same or different, and the second and third protein-binding motifs are the same or different.
The gene editing system of claim 76, further comprising

a second fusion protein comprising the first protease fragment and a second RNA binding domain, or a polynucleotide encoding the second fusion protein,

wherein the first protease fragment and the second RNA binding domain are optionally connected by a linker,

wherein the mgRNA further comprises a second protein-binding motif, and

wherein the second RNA binding domain binds to the second protein-binding motif.
The gene editing system of any one of claims 74-79, wherein the protease is a TEV protease, a TuMV protease, a PPV protease, a PVY protease, a ZIKV protease, or a WNV protease.
The gene editing system in claim 80, wherein the protease is a TEV protease comprising a sequence of SEQ ID NO: 205.
The gene editing system in claim 81, wherein the first TEV protease fragment comprises a sequence of SEQ ID NO: 206.
The gene editing system in any one of claims 74-82, wherein the nucleobase deaminase inhibitor is an inhibitory domain of a nucleobase deaminase.
The gene editing system in any one of claims 74-83, wherein the nucleobase deaminase inhibitor is an inhibitory domain of a cytidine deaminase.
The gene editing system in claim 84, wherein the inhibitory domain of a cytidine deaminase comprises an amino acid sequence of SEQ ID NO: 222 or SEQ ID NO: 223.
The gene editing system in any one of claims 73-85, wherein the nucleotide deaminase is a cytidine deaminase.
The gene editing system in claim 86, wherein the cytidine deaminase is selected from the group consisting of APOBEC3B (A3B) , APOBEC3C (A3C) , APOBEC3D (A3D) , APOBEC3F (A3F) , APOBEC3G (A3G) , APOBEC3H (A3H) , APOBECI (Al) , APOBEC3 (A3) , APOBEC2 (A2) , APOBEC4 (A4) , and AICDA (AID) .
The gene editing system in claim 86, wherein the cytidine deaminase is a human or mouse cytidine deaminase.
The gene editing system in claim 88, wherein the catalytic domain of the cytidine deaminase is a mouse A3 cytidine deaminase domain 1 (mA3-CDAl) or human A3B cytidine deaminase domain 2 (hA3B-CDA2) .
The gene editing system in any one of claims 73-85, wherein the nucleotide deaminase is an adenosine deaminase.
The gene editing system in claim 90, wherein the adenosine deaminase is selected from the group consisting of tRNA-specific adenosine deaminase (TadA) , adenosine deaminase tRNA specific 1 (ADAT1) , adenosine deaminase tRNA specific 2 (ADAT2) , adenosine deaminase tRNA specific 3 (ADAT3) , adenosine deaminase RNA specific B1 (ADARB1) , adenosine deaminase RNA specific B2 (ADARB2) , adenosine monophosphate deaminase 1 (AMPD1) , adenosine monophosphate deaminase 2 (AMPD2) , adenosine monophosphate deaminase 3 (AMPD3) , adenosine deaminase (ADA) , adenosine deaminase 2 (ADA2) , adenosine deaminase like (ADAL) , adenosine deaminase domain containing 1 (ADAD1) , adenosine deaminase domain containing 2 (ADAD2) , and adenosine deaminase RNA specific (ADAR) .
The gene editing system of any one of claims 73-91, wherein the first fusion protein further comprises an uracil glycosylase inhibitor (UGI) .
The gene editing system of any one of claims 73-92, wherein the Cas protein is a Cas9, a dead Cas9 (dCas9) , or a Cas9 nickase (nCas9) selected from the group consisting of SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpfl, LbCpfl, FnCpfl, VQR Cas9, EQR Cas9, VRER Cas9, Cas9-NG, xCas9, eCas9, SpCas9-HF1, HypaCas9, HiFiCas9, sniper-Cas9, SpG, SpRY, KKH SaCas9, CjCas9, Cas9-NRRH, Cas9-NRCH, Cas9-NRTH, SsCpfl, PcCpfl, BpCpfl, LiCpfl, PmCpfl, Lb2Cpf1, PbCpfl, PbCpfl, PeCpf1, PdCpf1, MbCpf1, EeCpf1, CmtCpf1, BsCpfl, BhCasl2b, AkCasl2b, BsCasl2b, AmCasl2b, AaCasl2b, RfxCasl3d, LwaCasl3a, PspCasl3b, PguCasl3b, and RanCasl3b.
The gene editing system of any one of claims 73-93, wherein the first protein-binding RNA motif and the first RNA binding domain, the second protein-binding RNA motif and the second RNA binding domain, and the third protein-binding RNA motif and the third RNA binding domain, are each independently selected from the group consisting of a MS2 phage operator stem-loop and MS2 coat protein (MCP) or an RNA-binding section thereof,

a BoxB and N22P or an RNA-binding section thereof,

a telomerase Ku binding motif and Ku protein or an RNA-binding section thereof,

a telomerase Sm7 binding motif and Sm7 protein or an RNA-binding section thereof,

a PP7 phage operator stem -loop and PP7 coat protein (PCP) or an RNA-binding section thereof,

a SfMu phage Com stem-loop and Com RNA binding protein or an RNA-binding section thereof, and

a non-natural RNA aptamer and corresponding aptamer ligand or an RNA-binding section thereof.
The gene editing system of any one of claims 1-94, wherein the mgRNA and/or the hgRNA comprises a dual-RNA structure.
The gene editing system of claim 95, wherein the dual-RNA structure is formed by a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) , wherein the crRNA comprises the spacer.
The gene editing system of claim 95 or 96, wherein the mgRNA comprises a mcrRNA and a first tracrRNA, and the mcrRNA comprises the mgRNA spacer, wherein the hgRNA comprises a hcrRNA and a second tracrRNA, and the hcrRNA comprises the hgRNA spacer, and wherein the first tracrRNA and the second tracrRNA are same or different.
The gene editing system of claim 97, wherein the mcrRNA and the hcrRNA are

a. SEQ ID NO. 639 and SEQ ID NO: 647, respectively; or

b. SEQ ID NO. 640 and SEQ ID NO: 648, respectively; or

c. SEQ ID NO. 641 and SEQ ID NO: 649, respectively; or

d. SEQ ID NO. 642 and SEQ ID NO: 650, respectively; or

e. SEQ ID NO. 643and SEQ ID NO: 651, respectively; or

f. SEQ ID NO. 644 and SEQ ID NO: 652, respectively; or

g. SEQ ID NO. 645 and SEQ ID NO: 653, respectively; or

h. SEQ ID NO. 646 and SEQ ID NO: 654, respectively.
The gene editing system of any one of claims 96-98, wherein the tracrRNA is SEQ ID NO: 655.
A polynucleotide encoding the mgRNA and/or hgRNA in at least one of claims 1-72.
A polynucleotide encoding all components except the first and second Cas proteins in the gene editing system in any one of claims 73-99.
A kit comprising, the polynucleotide in claim 101, and a polynucleotide encoding the first and/or second Cas protein in any one of claims 73-99.
A vector comprising the polynucleotide in claim 100.
A vector comprising the polynucleotide in claim 101.
The vector of any one of claims 103-104, wherein the vector is a plasmid or a viral vector.
The vector of any one of claims 103-105, wherein the vector is a polycistronic vector.
A kit comprising

a. the vector in claim any one of claim 103-106,

b. a vector comprising the polynucleotide encoding the first and/or second Cas protein in any one of claims 73-99.
A cell comprising the gene editing system in any one of claims 1-99.
A cell comprising the polynucleotide in any one of claims 100-101.
The cell in claim 109, further comprising a polynucleotide encoding the first and/or second Cas protein in any one of claims 73-99.
A cell comprising the vector in any one of claims 103-106.
The cell in claim 111, further comprising a vector comprising a polynucleotide encoding the first and/or second Cas protein in any one of claims 73-99.
The cell of any one of claims 108-112, wherein the cell is infected by HBV.
The cell of any one of claims 108-113, wherein the cell comprises an HBV covalently closed circular DNA (cccDNA) .
The cell of any one of claims 108-114, wherein the cell comprises an HBV integrated DNA.
The cell of any one of claims 108-115, wherein the cell is a liver cell.
The cell of any one of claims 108-116, wherein the cell is a hepatocyte.
A composition comprising the gene editing system in any one of claims 1-99.
A composition comprising the cell in any one of claims 108-117.
A method for disrupting an HBV gene in a cell, comprising introducing into the cell the gene editing system in any one of claims 1-99.
A method for disrupting an HBV S gene in a cell, comprising introducing into the cell the gene editing system in any one of claims 1-2, 11-12, 17-18, 23-24, 29-30, 35-36, 41-42, 47-48, 53-54, and 59-60.
A method for disrupting an HBV PreS1 gene in a cell, comprising introducing into the cell the gene editing system in any one of claims 3-4, 13-14, 19-20, 25-26, 31-32, 37-38, 43-44, 49-50, 55-56, and 61-62.
A method for disrupting an HBV P gene in a cell, comprising introducing into the cell the gene editing system in claim 5 or 6.
A method for disrupting an HBV X gene in a cell, comprising introducing into the cell the gene editing system in claim 7 or 8.
A method for disrupting an HBV C gene in a cell, comprising introducing into the cell the gene editing system in any one of claims 9-10, 15-16, 21-22, 27-28, 33-34, 39-40, 45-46, 51-52, 57-58, and 63-64.
A method for inducing a missense mutation in an HBV gene, comprising introducing into the cell the gene editing system in any one of claims 66-70.
A method for targeting an NTCP receptor of a cell, comprising introducing into the cell the gene editing system of claim 71 or 72.
A method for treating HBV infection in a subject, comprising disrupting an HBV gene in a cell of the subject with any one of the methods in claims 120-127.
A method for treating or preventing chronic hepatitis B (CHB) , liver cirrhosis, hepatocellular carcinoma, or liver failure comprising disrupting an HBV gene in a cell of the subject with any one of the methods in claims 120-128.
The method in claims 120-129, wherein the cell is infected by HBV.
The method in claims 120-130, wherein the cell comprises an HBV covalently closed circular DNA (cccDNA) .
The method in claims 120-131, wherein the cell comprises an HBV integrated DNA.
The method in claims 120-132, wherein the cell is a liver cell.
The method in claims 120-133, wherein the cell is a hepatocyte.