Systems and methods for large fragment nucleic acid deletion

Abstract

Compositions and methods for deleting a target DNA sequence from a target DNA molecule are provided. The disclosed GRAND-Del system employs a pair of pegRNAs that target nearby genomic sites and have mutually complementary sequences to jointly form a template for inserting an exogenous sequence into a target DNA molecule to replace a target DNA sequence to be deleted. The deleted chromosomal DNA sequence can be degraded, randomly inserted (resulting in random transposon), or inserted at a target location, such as at different locations of the same chromosome or in different chromosomes (resulting in targeted transposon) or in centromere-containing plasmids (resulting in the production of artificial chromosomes).

Description

Technical Field Background Technology

[0002] Targeted deletion of genome sequences is a useful method for generating gene knockouts. Gene knockout allows researchers to study the function of specific genes in vivo and understand their role in normal development and physiology, as well as disease pathology. By studying the phenotype of organisms with knockout genes, researchers can gain deeper insights into the biological processes involved by that gene.

[0003] Targeted deletion also has clinical significance. Gene duplication, or chromosomal duplication, or gene amplification, is a major mechanism for the generation of new genetic material during molecular evolution. Gene duplication may arise as a product of several types of errors in DNA replication and repair mechanisms, or it may arise through accidental capture by selfish genetic elements.

[0004] Duplication of oncogenes is a common cause of many types of cancer. In this case, the genetic duplication occurs in somatic cells, affecting only the genome of the cancer cell itself, without affecting the entire organism. Recent comprehensive patient-level classification and quantification of driving events in the TCGA cohort showed that there were an average of 12 driving events per tumor, of which 1.5 were amplifications of oncogenes.

[0005] Erroneous gene duplications can also lead to other diseases. For example, gene duplications occur in approximately 10% of patients with Duchenne muscular dystrophy (DMD) / Becker muscular dystrophy (BMD). Furthermore, DMD can be treated by deleting parts of the mutated gene.

[0006] Peroneal muscular atrophy type 1A (CMT1A) is caused by a duplication of the PMP22 gene on chromosome 17, leading to an overproduction of peripheral nerve myelin 22 (PMP22). CMT1A is a neurological disorder characterized by progressive muscle weakness and atrophy, primarily affecting the peripheral nerves.

[0007] Hereditary stress-prone peripheral neuropathy (HNPP) is another type of peripheral neuropathy caused by deletion or duplication of the 17p11.2 region (including the PMP22 gene). HNPP leads to nerve damage and recurrent episodes of weakness or numbness, usually triggered by stress or trauma.

[0008] Some cases of familial amyotrophic lateral sclerosis (FALS) have been associated with gene duplication. For example, superoxide dismutase 1 (SOD1) SOD1 Duplication of the gene (encoding an enzyme involved in antioxidant defense) can lead to FALS. Mutations... SOD1 Excessive copying of genes leads to the accumulation of toxic proteins, which in turn causes degeneration of motor neurons.

[0009] 15q duplication syndrome is a chromosomal disorder caused by duplication of a segment of chromosome 15. It can lead to various developmental delays, intellectual disabilities, seizures, and autism spectrum disorders. The duplication may involve different regions of chromosome 15, such as the proximal 15q11-q13 region, resulting in different clinical features.

[0010] Similarly, 17p duplication syndromes involve duplication of a segment of the 17p chromosome, which can lead to syndromes characterized by intellectual disability, developmental delay, and distinctive facial features. Chromosomal-scale deletions may have the potential to treat Down syndrome (which has an extra chromosome 21).

[0011] However, targeted and specific gene deletion, or more broadly, genomic sequence deletion, remains challenging. CRISPR-Cas9-based deletion methods have been reported, but they can only delete very small fragments with sufficient efficiency and are prone to errors, including small insertions and deletions as well as unexpected deletions. Small insertions and deletions can activate the p53 pathway, leading to unexpected adverse reactions in patients.

[0012] There is still a pressing need for improved methods to achieve targeted and specific gene deletion, including the deletion of large segments of the genome. Summary of the Invention

[0013] Efficient targeted deletion holds immense potential for treating a variety of genetic diseases, such as cancer and DMD / BMD. This is also crucial for scientific research, such as elucidating the function of genes or non-coding elements and constructing disease models.

[0014] The various embodiments described herein provide compositions and methods for deleting target DNA sequences from target DNA molecules. The disclosed GRAND-Del system employs a pair of leader editing guide RNAs (pegRNAs) that target nearby genomic sites (whether linearly or spatially, e.g., in three or four dimensions) and have complementary sequences to form a template for inserting a foreign sequence into the target DNA molecule to replace the target DNA sequence to be deleted.

[0015] One embodiment of this disclosure provides a method for deleting a target DNA sequence between a first position and a second position in a target DNA molecule, the method comprising contacting the target DNA molecule with: (a) a Cas protein and reverse transcriptase, (b) a first lead editing guide RNA (pegRNA) comprising a first primer binding site (PBS), a first spacer sequence, and a first reverse transcriptase template (RTT), and (c) a second pegRNA comprising a second PBS, a second spacer sequence, and a second RTT, wherein (i) the first RTT comprises a first fragment and a first paired fragment, (ii) the second RTT comprises a second fragment and a second paired fragment, (iii) the first paired fragment and the second paired fragment are complementary to each other, and (iv) the first fragment and the second fragment each have a length of 0-2000 nt, wherein (1) the first spacer sequence and the first PBS target the first pegRNA to the first position, allowing the Cas protein and reverse transcriptase to create a nick at the first position and extend the first strand of the target DNA molecule using the first RTT as a template to form a first new single strand complementary to the first RTT, and (2) The second spacer sequence and the second PBS target the second pegRNA to the second position, allowing the Cas protein and reverse transcriptase to create a nick at the second position and use the second RTT as a template to extend the second strand of the target DNA molecule to form a second new single strand complementary to the second RTT, wherein the first new single strand and the second new single strand form a double-stranded region, displacing the target DNA sequence from the target DNA molecule.

[0016] In some embodiments, the first and second paired segments each have a length of 2-2000 nt, preferably 20-60 nt, and more preferably 25 to 40 nt.

[0017] In some implementations, the target DNA sequence has a length of at least 10 bp, 100 bp, 1 Kb, 10 Kb, 100 Kb, 1 Mb, 10 Mb, 20 Mb, 30 Mb, 40 Mb, 50 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb, or 500 Mb.

[0018] In some implementations, the first segment and the second segment each have a length of 10-1000 nt, or 10-500 nt, or 10-100 nt.

[0019] In some implementations, the first fragment and the second fragment each independently have sequence complementarity of less than 95%, or less than 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% with the target DNA sequence.

[0020] In some implementations, the Cas protein is a nickase. In some embodiments, the cleavage enzyme is a Cas9 protein containing an inactivated HNH domain that cleaves the target chain, wherein the Cas9 protein is optionally selected from SpyCas9, SauCas9, NmeCas9, StCas9, FnCas9, CjCas9, AnaCas9, and GeoCas9, or wherein the Cas protein is a Cas12 protein, optionally selected from Cas12a, Cas12b, Cas12f, and Cas12i, further wherein the Cas12 protein is optionally selected from AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b, and LsCas12b.

[0021] In some implementations, the reverse transcriptase is an M-MLV reverse transcriptase or a reverse transcriptase capable of functioning under physiological conditions.

[0022] In some embodiments, the nicking enzyme and reverse transcriptase are provided as nucleotides encoding the respective proteins, or as proteins themselves.

[0023] In some implementations, each pegRNA is provided as recombinant DNA encoding the pegRNA or as an RNA molecule.

[0024] In some implementations, the target DNA molecule is a chromosome, a chromosome fragment, or circular DNA.

[0025] In some embodiments, the method further includes contacting a target DNA sequence removed from a target DNA molecule with a receiving DNA molecule, the receiving DNA molecule comprising two single-stranded ends complementary to at least a portion of the two strands of the target DNA sequence.

[0026] In some implementations, the receiving DNA molecule is edited by one or more leader editors to introduce the chromosome at both single-stranded ends.

[0027] In some embodiments, the receiving DNA is a plasmid. In some embodiments, the plasmid includes a centromere.

[0028] In some embodiments, the method further includes creating nicks at one or more sites on the target DNA molecule, preferably within the target DNA sequence or within 1000 nt from the target DNA sequence.

[0029] In some embodiments, the contact takes place in mammalian cells, prokaryotic cells, eukaryotic cells, or plant cells. In some embodiments, the contact takes place in vitro, ex vivo, or in vivo. In some embodiments, the contact takes place in a human patient. In some embodiments, the human patient suffers from a disease mediated by a gene duplication or defective gene.

[0030] In some implementations, the duplicated or defective gene is selected from the following genes: oncogenes, dystrophin. PMP22 Gene at chromosome locus 17p11.2, superoxide dismutase 1 (SOD1) SOD1 Genes at chromosome loci 15q11-q13, genes at locus 17p, and β-globin genes.

[0031] In some embodiments, compositions or kits for deleting a target DNA sequence between a first position and a second position in a target DNA molecule are also provided, comprising: (a) a first lead editing guide RNA (pegRNA) including a first primer binding site (PBS), a first spacer sequence, and a first reverse transcriptase template (RTT); and (b) a second pegRNA including a second PBS, a second spacer sequence, and a second RTT, wherein (i) the first RTT includes a first fragment and a first paired fragment, (ii) the second RTT includes a second fragment and a second paired fragment, (iii) the first paired fragment and the second paired fragment are complementary to each other, and (iv) the first fragment and the second fragment each have a length of 0-2000 nt, wherein (1) the first spacer sequence and the first PBS can target the first pegRNA to the first position, allowing the Cas protein and reverse transcriptase to create a nick at the first position and extend the first strand of the target DNA molecule using the first RTT as a template to form a first new single strand complementary to the first RTT, and (2) The second spacer sequence and the second PBS target the second pegRNA to the second position, allowing the Cas protein and reverse transcriptase to create a nick at the second position and use the second RTT as a template to extend the second strand of the target DNA molecule to form a second new single strand complementary to the second RTT, wherein the first new single strand and the second new single strand can form a double-stranded region to displace the target DNA sequence from the target DNA molecule.

[0032] In some embodiments, the composition or kit further includes Cas protein and reverse transcriptase.

[0033] Also provided are one or more polynucleotides for deleting a target DNA sequence between a first position and a second position in a target DNA molecule, comprising or encoding: (a) a first lead editing guide RNA (pegRNA) comprising a first primer binding site (PBS), a first spacer sequence, and a first reverse transcriptase template (RTT), and (b) a second pegRNA comprising a second PBS, a second spacer sequence, and a second RTT, wherein (i) the first RTT comprises a first fragment and a first paired fragment, (ii) the second RTT comprises a second fragment and a second paired fragment, (iii) the first paired fragment and the second paired fragment are complementary to each other, and (iv) the first fragment and the second fragment each have a length of 0-2000 nt, wherein (1) the first spacer sequence and the first PBS can target the first pegRNA to the first position, allowing the Cas protein and reverse transcriptase to create a nick at the first position and extend the first strand of the target DNA molecule using the first RTT as a template to form a first new single strand complementary to the first RTT, and (2) The second spacer sequence and the second PBS target the second pegRNA to the second position, allowing the Cas protein and reverse transcriptase to create a nick at the second position and use the second RTT as a template to extend the second strand of the target DNA molecule to form a second new single strand complementary to the second RTT, wherein the first new single strand and the second new single strand can form a double-stranded region to displace the target DNA sequence from the target DNA molecule.

[0034] In some embodiments, the one or more polynucleotides further include or encode Cas proteins and reverse transcriptases. Attached Figure Description

[0035] Figure 1 This explains the components and editing process of the GRAND-Del editor.

[0036] Figure 2 The process of verifying GRAND-Del mediated deletion results is demonstrated.

[0037] Figure 3 The method and results for testing GRAND-Del efficiency for targeted sequences ranging in length from 10 Kb to 100 Mb are shown.

[0038] Figure 4 The method and results for measuring the accuracy of deleted connection points are shown.

[0039] Figure 5 This demonstrates an alternative design for the GRAND-Del editor and its impact on editing efficiency.

[0040] Figure 6This demonstrates that the GRAND-Del technology can be applied to multiple operations at multiple sites with high efficiency.

[0041] Figure 7 This shows the destination of the chromosome fragments released from the GRAND-Del operation.

[0042] Figure 8 Visualization of chromosome fragments released by GRAND-Del in HEK293T cells is shown.

[0043] Figure 9 The design and validation of the targeted transposal are shown.

[0044] Figure 10 The study showed that introducing sgRNA-induced nicks improved targeted transposition.

[0045] Figure 11 This explains the construction of artificial chromosomes. Detailed Implementation

[0046] Technical terms: It should be noted that the terms "an" or "a" entity refer to one or more of the same entity; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "an" (or "a"), "one or more," and "at least one" are used interchangeably in this document.

[0047] As used herein, the term "peptide" is intended to encompass both the singular and plural "peptide" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "peptide" refers to any chain or multiple chains of two or more amino acids, and does not refer to a product of a specific length. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or multiple chains of two or more amino acids are included within the definition of "peptide," and the term "peptide" may be used in place of any of these terms, or used interchangeably with them. The term "peptide" is also intended to refer to products modified after peptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. Peptides may be derived from natural biological sources or produced through recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be produced in any way, including through chemical synthesis.

[0048] The term "encoding" applied to polynucleotides refers to a polynucleotide that, if it can be transcribed and / or translated in its natural state or when manipulated by methods well known to those skilled in the art, "encodes" the polypeptide. The antisense strand is the complementary strand of this nucleic acid, and the coding sequence can be deduced from it.

[0049] The term "Cas protein" or "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated (Cas) Protein" refers to RNA-guided DNA endonucleases associated with the CRISPR adaptive immune system in Streptococcus pyogenes and other bacteria. Cas proteins include Cas9, Cas12a (Cpf1), Cas12b (formerly known as C2c1), Cas13, 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, and RHA. FnCas9, KKHSaCas9, NmeCas9, StCas9, CjCas9, AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, P b4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b, LsCas12b, RfCas13d, LwaCas13a, PspCas13b, PguCas13b, RanCas13b.

[0050] GRAND deletion (GRAND-Del) This invention discloses a novel gene editing method called GRAND (genome editing via RTT double pegRNAs that are at least partially aligned with each other but not homologous to the target sequence) deletion, or simply GRAND-Del, which is capable of deleting the target genome sequence.

[0051] The exemplary GRAND-Del technology employs Figure 1The image shows a pair of guide RNA (pegRNA) molecules. Traditional pegRNAs, in addition to CRISPR RNA (crRNA), which can be provided as a single guide RNA (sgRNA) along with trRNA, include a reverse transcriptase (RT) template (RTT) and a primer binding site (PBS). The PBS is complementary to the guide sequence (or "spacer sequence") in the sgRNA, but is typically a few nucleotides shorter. When the guide sequence binds to the target genome sequence and dissociates the DNA double helix, the PBS binds to the opposite strand and uses the RTT as a template to initiate reverse transcription. The RTT can contain mutations or small insertions relative to the target genome sequence, but needs to be largely homologous to the target genome sequence.

[0052] In each of the two pegRNAs in the GRAND-Del system, the RTT does not necessarily have to be homologous to the target genomic sequence. In some implementations, the RTT preferably has reduced or even no homology to the target genomic sequence. Instead, the two RTTs (RTT1 and RTT2 as shown in the figure) share a complementary portion. For example, as Figure 1 As shown, the RTT1 of pegRNA1 and the RTT2 of pegRNA2 have complementary sequences (or substantially complementary, for example, at least 40%, 60%, 70%, 80%, 90%, or 95% complementary sequence identity), which allows them (or their products) to pair with each other.

[0053] like Figure 1 As shown, pairing does not occur between the two pegRNA molecules. Instead, after binding to the target genomic sequence (steps 100 and 110), both pegRNAs serve as templates (RTT1 and RTT2) to produce (via reverse transcription) DNA sequences (201 and 202; single-stranded) (step 120). Figure 1 As shown in step 130, by means of complementary sequences and their close proximity, the two newly reverse-transcribed single-stranded DNA fragments (201 and 202, also known as “flaps”) can bind together (step 120) to form a double-stranded region, replacing the original genomic sequences (101 and 102), which are typically much longer. The replaced genomic sequence can be degraded or digested by enzymes in the cell. Simultaneously, the newly formed double strands are integrated into the genome through DNA repair (step 120).

[0054] In the alternative design, each of RTT1 and RTT2 also includes a proximal region (PR) between the complementary distal region and PBS. Figure 5a). Therefore, the newly generated lobes hybridize (“overlap”) at the distal ends, but leave two single-stranded regions (the products of PR1 and PR2) in the target genome. These single-stranded regions can serve as templates for DNA replication, and the final inserted sequence (to replace the deleted genomic sequence) includes PR1, the overlapping portion, and PR2 (and its complementary portion).

[0055] As demonstrated in experimental examples, GRAND-Del technology is highly efficient (e.g., >50% efficiency for target sequences up to 10 kb long) and can delete target sequences up to 100 Mb. Equally important, unlike existing technologies, GRAND-Del does not produce dense DNA double-strand breaks (DSBs), thus avoiding unwanted byproducts such as insertions, deletions, and inversions.

[0056] Compared to methods that rely on DSB, GRAND-Del technology may have a lower risk of p53 activation. GRAND-Del limits off-target editing by using a Cas9 nickase and paired pegRNAs with RTTs that are not homologous to the target sequence. In contrast, existing technologies lead to p53 activation.

[0057] Therefore, one embodiment of this disclosure provides a method for deleting a target DNA sequence between a first position and a second position in a target DNA molecule, the method comprising contacting the target DNA molecule with: (a) a Cas protein (e.g., a conventional Cas9, Cas12, or Cas13 protein, or a nicking enzyme) and a reverse transcriptase (optionally bound to a fusion protein, or provided separately), (b) a first lead editing guide RNA (pegRNA) comprising a first primer binding site (PBS), a first spacer sequence, and a first reverse transcriptase template (RTT), and (c) a second pegRNA comprising a second PBS, a second spacer sequence, and a second RTT.

[0058] In some implementations, (i) the first RTT includes a first segment and a first paired segment, (ii) the second RTT includes a second segment and a second paired segment, (iii) the first paired segment and the second paired segment are complementary to each other, and (iv) the first segment and the second segment each have a length of 0-2000 nt. In other words, the first segment and the second segment are each optional.

[0059] In some implementations, (1) a first spacer sequence and a first PBS target the first pegRNA to a first position, allowing the Cas protein and reverse transcriptase to create a nick at the first position and use the first RTT as a template to extend the first strand of the target DNA molecule to form a first new single strand complementary to the first RTT, and (2) a second spacer sequence and a second PBS target the second pegRNA to a second position, allowing the Cas protein and reverse transcriptase to create a nick at the second position and use the second RTT as a template to extend the second strand of the target DNA molecule to form a second new single strand complementary to the second RTT.

[0060] In some implementations, a first new single strand and a second new single strand form a double-stranded region, displacing the target DNA sequence from the target DNA molecule.

[0061] In summary, the inverse complementary sequences of the first fragment, the first paired fragment, and the second fragment encode one strand of the nucleic acid sequence to be inserted into the target DNA molecule. It should be noted that both the first and second fragments can be empty (0 nucleotides) or thousands of nucleotides long. Simultaneously, the target DNA sequence (between the first and second positions on the target DNA molecule) is substituted and deleted. Typically, as demonstrated, the newly inserted fragment can be very short (e.g., <100 bp), while the deleted target DNA sequence can be quite long (e.g., hundreds of Mb).

[0062] In some embodiments, the first and second paired segments each have a length of less than 200 nt, or less than 150 nt, less than 120 nt, less than 100 nt, less than 80 nt, less than 70 nt, less than 60 nt, less than 50 nt, less than 40 nt, or less than 35 nt. In some embodiments, the first and second paired segments each have a length of 5-100 nt, 10-60 nt, 20-50 nt, or 25 to 40 nt.

[0063] The first fragment (if included), one of the paired fragments, and the second fragment (if included, their reverse complementary sequences) together encode a nucleic acid sequence to be inserted into the target genome sequence to replace and delete the target DNA sequence. In some embodiments, the total coding sequence is at least 10 bp in length, or at least 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90, or 100 bp. In some embodiments, the total coding sequence is not longer than 1 kb, or not longer than 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, or 30 bp.

[0064] In some embodiments, the length of each of the first and second segments is at least 0 bp, or at least 2 bp, 5 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, or 100 bp. In some embodiments, the length of each of the first and second segments is not longer than 1 Kb, or not longer than 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, or 30 bp.

[0065] As disclosed, the first fragment, the second fragment, and / or the paired fragments do not need to be homologous to the genomic sequence to be replaced. In some embodiments, the first fragment and the second fragment each independently have less than 95%, or less than 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% sequence complementarity with the target DNA sequence.

[0066] As illustrated in the examples, the target DNA sequence (to be deleted) may be quite long. In some implementations, the target DNA sequence has a length of at least 10 bp, 100 bp, 1 Kb, 10 Kb, 100 Kb, 1 Mb, 10 Mb, 20 Mb, 30 Mb, 40 Mb, 50 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb, or 500 Mb.

[0067] The pegRNA disclosed in this paper is similar to the conventional pegRNA used in lead editing. Lead editing is a genome editing technique that modifies the genome of an organism. Lead editing directly writes new genetic information into a target DNA site. It uses a fusion protein, formed by fusing a catalytically impaired endonuclease (e.g., Cas9) with an engineered reverse transcriptase, and a lead editing guide RNA (pegRNA) that recognizes the target site and provides new genetic information to replace the target DNA nucleotides. Lead editing mediates targeted insertions, deletions, and base-to-base conversions without requiring DNA double-strand breaks (DSBs) or donor DNA templates.

[0068] pegRNAs recognize the target nucleotide sequence to be edited and encode new genetic information to replace the target sequence. A pegRNA consists of an extended single-guide RNA (sgRNA) (or just crRNA) containing a primer binding site (PBS) and a reverse transcriptase (RT) template. During genome editing, the primer binding site allows the 3' end of the cleaved DNA strand to hybridize with the pegRNA, while the RTT serves as a template for synthesizing the edited genetic information. Within the sgRNA or crRNA portion, there is a spacer sequence (guide sequence) that guides the lead editor to the target genomic site, and an sgRNA / crRNA scaffold.

[0069] In some embodiments, the fusion protein includes a nicking enzyme fused with a reverse transcriptase. The nicking enzyme can be derived from a conventional Cas9 protein, such as SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpf1, FnCpf1, VQR SpCas9, EQR SpCas9, VRER SpCas9, SpCas9-NG, xSpCas9, RHA FnCas9, KKHSaCas9, NmeCas9, StCas9, or CjCas9. An exemplary nicking enzyme is Cas9 H840A. The Cas9 enzyme contains two nuclease domains capable of cleaving DNA sequences: a RuvC domain that cleaves the non-target strand and an HNH domain that cleaves the target strand. The HNH domain is inactivated by introducing an H840A substitution (where histidine at position 840 is replaced with alanine). Because only the RuvC functional domain catalyzes the introduction of a single-strand nick into the impaired Cas9, it is a nicking enzyme.

[0070] In some embodiments, the Cas protein is a Cas12 protein, such as Cas12a, Cas12b, Cas12f, and Cas12i. In some embodiments, the Cas12 protein is selected from AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b, and LsCas12b.

[0071] Non-restricted examples of reverse transcriptases include human immunodeficiency virus (HIV) reverse transcriptase, Moloney mouse leukemia virus (M-MLV) reverse transcriptase, and avian myeloblastoma virus (AMV) reverse transcriptase, as well as any reverse transcriptase that can function under physiological conditions.

[0072] In some implementations, the lead editing system also includes a single-guide RNA (sgRNA) (or crRNA only) that guides the Cas9 H840A nickase portion of the fusion protein to create a nick on the non-edited DNA strand. However, it should be noted that the GRAND-Del system does not require this additional sgRNA / crRNA.

[0073] Leader editing can be performed by transfecting target cells with pegRNA and fusion proteins. Transfection is typically accomplished by introducing a vector into the cell. In some implementations, leader editors can be introduced directly into cells as plasmids, linear DNA, proteins, RNA, and virus-like particles or complexes thereof. Each molecule can be introduced individually or together; there are no restrictions.

[0074] Vectors can be introduced into desired host cells using known methods, including but not limited to transfection, transduction, cell fusion, and liposome transfection. Vectors may include various regulatory elements, including promoters. In some embodiments, this disclosure provides expression vectors comprising any of the polynucleotides described herein, for example, expression vectors comprising polynucleotides encoding fusion proteins and / or pegRNAs.

[0075] Spacer sequences and primer binding sites can be designed to bind to genomic sequences flanking the regions where DNA insertion and / or replacement is desired.

[0076] Therefore, in some embodiments, the first pegRNA further includes a first primer binding site (PBS) and a first spacer sequence, enabling the fusion protein or complex to reverse transcribe the first template sequence at a first PBS target sequence near the target site, wherein the first PBS target sequence is complementary to the first PBS; the second pegRNA includes a second PBS and a second spacer sequence, enabling the fusion protein or complex to reverse transcribe the second template sequence at a second PBS target sequence near the target site, wherein the second PBS target sequence is complementary to the second PBS. In some embodiments, the first RTT and the second RTT are reverse transcribed, thereby causing the first paired fragment generated by reverse transcription to pair with the second paired fragment generated by reverse transcription.

[0077] As illustrated in Examples 5-7, the target DNA sequence deleted from the target DNA molecule (also known as a "released" sequence, such as a released chromosomal fragment) can be randomly inserted into different locations on the chromosome or into different chromosomes. In some implementations, this integration can be targeted to a specific location, for example, by introducing a "lobe" (a single-stranded DNA fragment) at the target location.

[0078] For example, lobes can be introduced using conventional lead editing (PE) techniques that provide a reverse transcription template. When the two lobes introduced at the target site are complementary to the ends (or near the ends) of the released sequence, this complementarity can help initiate the integration process, allowing the released sequence to be inserted into the target site.

[0079] In some implementations, the target site is within the chromosome. This targeted insertion, along with earlier targeted deletion, leads to targeted transposition.

[0080] In some implementations, the target site is in a plasmid, such as a plasmid containing a centromere. When the released chromosome fragment is inserted into such a plasmid, it produces an artificial chromosome.

[0081] In some embodiments, to improve the efficiency of targeted transposition, one or more nick sites may be introduced at the target DNA molecule where the released fragment is generated. Nick sites may be generated, for example, by additional sgRNA. In some embodiments, there are 1, 2, 3, 4, 5, or more nick sites within the target sequence to be deleted. In some embodiments, the nick sites are located close to the deletion site, for example, within 5000 nt, 2000 nt, 1000 nt, 500 nt, 300 nt, 200 nt, 100 nt, or 50 nt of either end of the target sequence to be deleted. In some embodiments, the nick sites are on a single strand of the target DNA molecule. In some embodiments, the nick sites are on both strands of the target DNA molecule.

[0082] In some embodiments, the contact occurs in the presence of a DNA repair system that forms a double-stranded DNA sequence at the target site, wherein one strand of the double-stranded DNA sequence is encoded by a first fragment, a first paired fragment, and an inverse complementary sequence of a second fragment. This contact can occur, for example, in cells, in vitro, in vitro, or in vivo. Cells can be prokaryotic cells, eukaryotic cells, plant cells, animal cells, mammalian cells, or human cells.

[0083] In some embodiments, the method is performed in a human patient. In some embodiments, the human patient suffers from a disease mediated by gene duplication. Exemplary diseases and the gene duplications involved are known in the art.

[0084] In one implementation, the disease is cancer, and the duplicated gene is an oncogene. In another implementation, the disease is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), and the duplicated or defective gene is dystrophin, where deletion of part of the gene can restore protein function. In one implementation, the disease is peroneal muscular dystrophy type 1A (CMT1A), and the duplicated gene is... PMP22 .

[0085] In one implementation, the disease is hereditary stress-prone peripheral neuropathy (HNPP), with a duplicated gene located on chromosome 17p11.2, including... PMP22 In one implementation, the disease is familial amyotrophic lateral sclerosis (FALS), and the duplicated gene is superoxide dismutase 1 (SOD1). SOD1 )Gene.

[0086] In one embodiment, the disease is 15q duplication syndrome, where the duplicated gene is located in the 15q11-q13 region. In another embodiment, the disease is 17p duplication syndrome, where the duplicated gene is located in the 17p region.

[0087] In one embodiment, the disease is a hereditary hemoglobin persistence (HPFH)-like blood disorder, and the duplicated gene is a β-globin gene. In another embodiment, the disease is a trisomy disorder, and the duplicated gene is a gene or gene cluster that is misrepeated on a chromosome.

[0088] Compositions, kits, and packaging for GRAND-DEL are also provided. In some embodiments, the composition, kit, or packaging includes at least one pair of pegRNAs for editing, as described herein.

[0089] In one embodiment, the composition or kit is used to delete a target DNA sequence between a first position and a second position in a target DNA molecule, and includes (a) a first lead editing guide RNA (pegRNA) comprising a first primer binding site (PBS), a first spacer sequence, and a first reverse transcriptase template (RTT), and (b) a second pegRNA comprising a second PBS, a second spacer sequence, and a second RTT. In some embodiments, (i) the first RTT comprises a first fragment and a first paired fragment, (ii) the second RTT comprises a second fragment and a second paired fragment, (iii) the first paired fragment and the second paired fragment are complementary to each other, and (iv) the first fragment and the second fragment each have a length of 0-2000 nt. In some embodiments, (1) a first spacer sequence and a first PBS can target the first pegRNA to a first position, allowing the Cas protein and reverse transcriptase to create a nick at the first position and extend the first strand of the target DNA molecule using the first RTT as a template to form a first new single strand complementary to the first RTT, and (2) a second spacer sequence and a second PBS can target the second pegRNA to a second position, allowing the Cas protein and reverse transcriptase to create a nick at the second position and extend the second strand of the target DNA molecule using the second RTT as a template to form a second new single strand complementary to the second RTT, wherein the first new single strand and the second new single strand can form a double-stranded region, displacing the target DNA sequence from the target DNA molecule (while simultaneously deleting the target region). In some embodiments, the composition or kit further includes the Cas protein and reverse transcriptase.

[0090] As provided, one or more of pegRNA and / or Cas protein / reverse transcriptase may be provided as polynucleotides encoding them. Thus, in one embodiment, one or more polynucleotides are provided for deleting a target DNA sequence between a first position and a second position in a target DNA molecule, comprising or encoding (a) a first lead editing guide RNA (pegRNA) comprising a first primer binding site (PBS), a first spacer sequence and a first reverse transcriptase template (RTT), and (b) a second pegRNA comprising a second PBS, a second spacer sequence and a second RTT.

[0091] In some embodiments, (i) the first RTT comprises a first fragment and a first paired fragment, (ii) the second RTT comprises a second fragment and a second paired fragment, (iii) the first paired fragment and the second paired fragment are complementary to each other, and (iv) the first fragment and the second fragment each have a length of 0-2000 nt. In some embodiments, (1) the first spacer sequence and the first PBS can target the first pegRNA to a first position, allowing the Cas protein and reverse transcriptase to create a nick at the first position and extend the first strand of the target DNA molecule using the first RTT as a template to form a first new single strand complementary to the first RTT, and (2) the second spacer sequence and the second PBS can target the second pegRNA to a second position, allowing the Cas protein and reverse transcriptase to create a nick at the second position and extend the second strand of the target DNA molecule using the second RTT as a template to form a second new single strand complementary to the second RTT. In some embodiments, the first new single strand and the second new single strand can form a double-stranded region, displacing the target DNA sequence from the target DNA molecule. In some embodiments, the one or more polynucleotides further include or encode the Cas protein and the reverse transcriptase. Specific Implementation Example 1. Detection of GRAND-Del products This example tests a newly designed genome sequence targeting deletion method. This method is referred to as "GRAND-Del" in this paper. Figure 1 As shown.

[0093] In short, the GRAND-Del editor consists of a pair of pegRNAs and a Cas9 nickase-RT fusion protein. The paired pegRNAs guide the Cas9 nickase-RT to recognize the PAM sequence on the target chromosome and bind to and cleave the opposing target DNA strands, respectively. The 3' end of the nick site hybridizes to the corresponding PBS region of the pegRNA, and then the reverse transcriptase uses the RT template (RTT) to initiate and generate complementary new single-stranded DNA (ssDNA). The two newly generated ssDNAs anneal together and compete with the original genomic fragment. Through the DNA repair pathway, the original strand is cleaved and replaced by an insert introduced by the RTT.

[0094] To confirm successful deletion, PCR primers targeting the flanking region of the region to be deleted were selected. Figure 2 a). The regions to be deleted in the endogenous sites HPRT1 and VEGFA range in length from 10 Kb to 100 Mb, therefore they cannot be amplified by PCR. Only the deleted product sequence can be amplified. The amplified PCR products were further validated using Sanger sequencing. Figure 2 b). This example confirms that the target area was successfully deleted.

[0095] Example 2. Large fragment loss efficiency from 10 Kb to 100 Mb This example demonstrates the use of GRAND-Del in deleting genomic sequences ranging in length from 10 Kb to 100 Mb in the HEK293 cell genome.

[0096] Deletion efficiency was measured using droplet digital PCR (ddPCR). Two probes with different labels targeted a reference sequence (without deletion) and an edited sequence (with deletion), respectively. The reference sequence was located on a different chromosome than the edited site. The probes were designed to hybridize with the insert sequence (…). Figure 3 a). Missing efficiency is represented by insertion efficiency.

[0097] This method was used to quantify large deletion editing at four endogenous gene loci in HEK293T, including HPRT1 , VEGFA , LSP1 and HEK3 The result is as follows Figure 3 As shown in b. When the length of the sequence to be deleted is 10 Kb, the deletion efficiency is greater than 50% (at least for...). VEGFA and HPRT1 ).for HPRT1 , LSP1 and HEK3 When the length is 1 Mb or longer, the efficiency drops to about 5%. However, at the same length (1 Mb), VEGFA The efficiency at the missing point remained at approximately 40%.

[0098] At 10–30 Mb, the deletion efficiency at all sites was approximately 5% or less. However, even at 100 Mb, the GRAND-Del technique was still able to achieve... VEGFA and LSP1 The site produced a significant level of target deletion (1% or 2%).

[0099] Example 3. Checking for missing precision This example examines the targeting accuracy of the GRAND-Del method.

[0100] The accuracy of the targeted deletion (i.e., ligation site accuracy) in HEK293T cells was examined using next-generation sequencing (NGS). The ligation sequence was amplified by out-out PCR (using primers flanking the cleavage site). The purity of the correctly deleted product was shown in [data missing]. Figure 4 b in (V: VEGFA L: LSP1 The relevant sequences are shown in Figure 4 c As shown in the figure, the accuracy of a typical deletion of a 10 Kb sequence exceeds 99%. When the deleted sequence is 1 Mb in length, the accuracy is still as high as 97%, and when the deleted sequence is 100 Mb, the accuracy is as high as 93%.

[0101] Example 4. Lobe Optimization This example tests different designs using the GRAND-Del technique with different lobes.

[0102] exist Figure 1 In the example, the two lobes are 100% complementary to each other and form a complete double strand, replacing the sequence to be deleted. Figure 5 In section a, an alternative design is provided in which the lobes are only partially complementary. In other words, the two lobes share a complementary region (“overlap”) at their distal portions, but each includes an additional, non-complementary proximal region. In this alternative design, the final sequence inserted at the deletion site includes a first proximal region, a complementary region, and a second proximal region.

[0103] exist VEGFA The site assessment evaluated the impact of overlapping and non-complementary proximal region lengths, targeting deletion sequences of 1 Mb or 30 Mb. Figure 5 As shown in b and 5c, longer overlapping sequences (80 bases or longer) tend to reduce overall deletion efficiency, while the total length of the lobe has no significant effect.

[0104] The ability of GRAND-Del technology to handle multiple operations was also tested. Figure 6 In this study, GRAND-Del was applied to multiplex editing at multiple sites, and its efficiency was determined by ddPCR. As shown in the figure, the GRAND-Del technology uses 2 or 3 pairs of pegRNAs to perform 10 kb or 1 Mb deletions at different sites and exhibits high efficiency throughout the process.

[0105] Example 5. Fate of the GRAND-Del release fragment As shown above, PCR amplification confirmed that GRAND-Del successfully deleted a large chromosomal segment at the target site. In this example, whole-genome sequencing was used to investigate whether the deleted (or “released”) segment was actually removed.

[0106] Whole-genome sequencing (WGS) revealed the location (or deletion) of the 1 Mb release fragment from Chr 6 in HEK293T single-cell clones after GRAND-Del treatment. Results showed ( Figure 7 The released chromosome fragments have two primary outcomes and one secondary outcome. The primary outcome is degradation and transposition, and the secondary outcome is replication.

[0107] Figure 7c summarizes the event analysis at Chr 3, Chr 6, and Chr 17 in HEK293T single-cell clones after GRAND-Del treatment. N represents the number of single clones analyzed. As shown in the figure, a large portion of the released fragments transposonized to different locations in the genome.

[0108] Figure 8 This image shows a visualization of the released chromosome fragments using fluorescence in situ hybridization (FISH), specifically targeting a 100 Mb deletion on Chr 6. Column 1 (from left to right) represents the Chr 6 centromere (green), with a ~108 kb genomic sequence flanking the STAT6 gene labeled with a red fluorescent probe; the nucleus is indicated in blue. In column 1, the 100 Mb region was deleted and transferred to another chromosome. In column 2, the region released by GRAND-Del was duplicated. Column 3 shows the wild-type unedited chromosome, used as a control. White arrows indicate wild-type chromosomes. Abnormal chromosomes are indicated by arrows of the same color indicating the type of structural variation.

[0109] Similarly, in Figure 8 In (b), a FISH test was performed on the 1 Mb deletion on Chr 17. In column 1, the HER2 gene was tagged with a red fluorescent probe, and the 1 Mb region of Chr 17 was completely deleted. In column 2, the 1 Mb region of Chr 17 was not deleted. Column 3 shows the wild-type unedited chromosome, used as a control. In (a) or (b), Edit-1 and Edit-2 are different single-cell populations released from 100 Mb (a) or 1 Mb (b) samples from GRAND-Del treated samples, respectively.

[0110] Example 6. Targeted transposition for releasing chromosome fragments Based on the unexpected discovery that chromosome fragments released by GRAND-Del randomly reintegrate into chromosomes, the inventors envisioned that this reintegration could be specifically targeted. This example designs and tests such a specific targeted transposition method.

[0111] Figure 9 A diagram illustrates how a chromosome fragment released from chromosome A is captured by chromosome B, or by the same chromosome but at a different location, with the assistance of a PE-induced valve. The PE-induced valve is complementary to both ends of the released fragment. This capture is expected to be facilitated by DNA repair pathways.

[0112] The targeted transposition was confirmed by ligation PCR. Figure 9 b). Tris-acetic acid-EDTA (TAE) agarose gel analysis of PCR amplicon showed that the 1 Mb fragment released from Chr 6 or Chr 9 was integrated into HEK293T cells in both directions. LSP1 The left and right sides of the site are joined and amplified. For example... Figure 9 According to the summary in c, the 1 Mb fragment from Chr 6 integrates into HEK293T cells in two directions. LSP1 and HPRT1 The integration efficiency of the site can be as high as 1%.

[0113] Potential methods to further improve the efficiency of targeted transposition were also tested. In optimizing the design ( Figure 10 In (a), additional sgRNA-mediated nicks were introduced into chromosome A to obtain fragments containing sticky ends. Lobes on chromosome B, complementary to the sticky ends, captured the fragments released from chromosome A and facilitated their integration. The addition of these additional sgRNAs increased the integration of 1 Mb from Chr 6 into HEK293T cells. LSP1 Integration efficiency ( Figure 10 b), its efficiency was quantified by ddPCR.

[0114] Example 7. Construction of Artificial Chromosomes It is believed that chromosome fragments released from GRAND-Del operations can provide a basis for the preparation of artificial chromosomes.

[0115] like Figure 11 As shown, after releasing the chromosome fragment, a plasmid containing a centromere is added. This plasmid has been induced by PE to have two lobes complementary to the released fragment. After the released chromosome fragment comes into contact with the plasmid containing the centromere, an artificial chromosome can be constructed.

[0116] The scope of this disclosure is not limited to the specific embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

[0117] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. A method for deleting a target DNA sequence between a first position and a second position in a target DNA molecule, comprising contacting the target DNA molecule with: (a) a Cas protein and a reverse transcriptase, (b) a first lead editing guide RNA (pegRNA) comprising a first primer binding site (PBS), a first spacer sequence and a first reverse transcriptase template (RTT), and (c) a second pegRNA comprising a second PBS, a second spacer sequence and a second RTT, wherein (i) the first RTT comprises a first fragment and a first paired fragment, (ii) the second RTT comprises a second fragment and a second paired fragment, (iii) the first paired fragment and the second paired fragment are complementary to each other, and (iv) the first fragment and the second fragment each have a length of 0-2000 nt, wherein (1) the first spacer sequence and the first PBS target the first pegRNA to the first position, allowing the Cas protein and the reverse transcriptase to create a nick at the first position and extend the first strand of the target DNA molecule using the first RTT as a template to form a first new single strand complementary to the first RTT, and (2) The second spacer sequence and the second PBS target the second pegRNA to the second position, allowing the Cas protein and reverse transcriptase to create a nick at the second position and use the second RTT as a template to extend the second strand of the target DNA molecule to form a second new single strand complementary to the second RTT, wherein the first new single strand and the second new single strand form a double-stranded region, displacing the target DNA sequence from the target DNA molecule.

2. The method according to claim 1, wherein the first paired segment and the second paired segment each have a length of 2-2000 nt, preferably 20-60 nt, more preferably 25 to 40 nt.

3. The method according to claim 1 or 2, wherein the target DNA sequence has a length of at least 10 bp, 100 bp, 1 Kb, 10 Kb, 100 Kb, 1 Mb, 10 Mb, 20 Mb, 30 Mb, 40 Mb, 50 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb or 500 Mb.

4. The method according to any of the preceding claims, wherein the first segment and the second segment each have a length of 10-1000 nt, or 10-500 nt, or 10-100 nt.

5. The method according to any one of the preceding claims, wherein the first fragment and the second fragment each independently have sequence complementarity of less than 95%, or less than 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% with the target DNA sequence.

6. The method according to any one of the preceding claims, wherein the Cas protein is a cleavage enzyme.

7. The method of claim 6, wherein the cleavage enzyme is a Cas9 protein containing an inactivated HNH domain that cleaves the target chain, wherein the Cas9 protein is optionally selected from SpyCas9, SauCas9, NmeCas9, StCas9, FnCas9, CjCas9, AnaCas9 and GeoCas9, or wherein the Cas protein is a Cas12 protein, optionally selected from Cas12a, Cas12b, Cas12f and Cas12i, further wherein the Cas12 protein is optionally selected from AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b and LsCas12b.

8. The method according to any one of the preceding claims, wherein the reverse transcriptase is an M-MLV reverse transcriptase or a reverse transcriptase capable of functioning under physiological conditions.

9. The method according to any one of the preceding claims, wherein the nicking enzyme and the reverse transcriptase are each provided as nucleotides encoding the respective proteins, or as proteins.

10. The method according to any one of the preceding claims, wherein each pegRNA is provided as recombinant DNA encoding the pegRNA or as an RNA molecule.

11. The method according to any of the preceding claims, wherein the target DNA molecule is a chromosome or a segment of a chromosome or circular DNA.

12. The method according to any one of the preceding claims further comprises contacting the target DNA sequence removed from the target DNA molecule with a receiving DNA molecule, said receiving DNA molecule comprising two single-stranded ends complementary to at least a portion of the two strands of the target DNA sequence.

13. The method of claim 12, wherein the receiving DNA molecule is edited by one or more leader editors to introduce a chromosome with two single-stranded lobes.

14. The method of claim 12, wherein the received DNA is a plasmid.

15. The method of claim 14, wherein the plasmid comprises a centromere.

16. The method according to any one of claims 12-15, further comprising creating a nick at one or more sites on the target DNA molecule, preferably within the target DNA sequence or within 1000 nt from the target DNA sequence.

17. The method according to any of the preceding claims, wherein the contact takes place in mammalian cells, prokaryotic cells, eukaryotic cells or plant cells.

18. The method according to any of the preceding claims, wherein the contact is performed in vitro, outside the body, or inside the body.

19. The method according to any of the preceding claims, wherein the contact is performed in a human patient.

20. The method of claim 19, wherein the human patient suffers from a disease mediated by a gene duplication or defective gene.

21. The method of claim 20, wherein the gene duplication or defective gene is selected from the following gene sites: oncogene, dystrophin, PMP22, gene at chromosome 17p11.2, superoxide dismutase 1 (SOD1), gene at chromosome 15q11-q13, gene at 17p, and β-globin gene.

22. A composition or kit for deleting a target DNA sequence between a first position and a second position in a target DNA molecule, comprising: (a) A first lead editing guide RNA (pegRNA) comprising a first primer binding site (PBS), a first spacer sequence, and a first reverse transcriptase template (RTT), and (b) a second pegRNA comprising a second PBS, a second spacer sequence, and a second RTT, wherein (i) the first RTT comprises a first fragment and a first paired fragment, (ii) the second RTT comprises a second fragment and a second paired fragment, (iii) the first paired fragment and the second paired fragment are complementary to each other, and (iv) the first fragment and the second fragment each have a length of 0-2000 nt, wherein (1) the first spacer sequence and the first PBS can target the first pegRNA to a first site, allowing the Cas protein and reverse transcriptase to create a nick at the first site and extend the first strand of the target DNA molecule using the first RTT as a template to form a first new single strand complementary to the first RTT, and (2) The second spacer sequence and the second PBS target the second pegRNA to the second position, allowing the Cas protein and reverse transcriptase to create a nick at the second position and use the second RTT as a template to extend the second strand of the target DNA molecule to form a second new single strand complementary to the second RTT, wherein the first new single strand and the second new single strand can form a double-stranded region to displace the target DNA sequence from the target DNA molecule.

23. The composition or kit according to claim 22 further comprises Cas protein and reverse transcriptase.

24. One or more polynucleotides for deleting a target DNA sequence between a first position and a second position in a target DNA molecule, comprising or encoding: (a) a first lead editing guide RNA (pegRNA) comprising a first primer binding site (PBS), a first spacer sequence, and a first reverse transcriptase template (RTT), and (b) a second pegRNA comprising a second PBS, a second spacer sequence, and a second RTT, wherein (i) the first RTT comprises a first fragment and a first paired fragment, (ii) the second RTT comprises a second fragment and a second paired fragment, (iii) the first paired fragment and the second paired fragment are complementary to each other, and (iv) the first fragment and the second fragment each have a length of 0-2000 nt, wherein (1) the first spacer sequence and the first PBS can target the first pegRNA to the first position, allowing the Cas protein and reverse transcriptase to create a nick at the first position and extend the first strand of the target DNA molecule using the first RTT as a template to form a first new single strand complementary to the first RTT, and (2) The second spacer sequence and the second PBS target the second pegRNA to the second position, allowing the Cas protein and reverse transcriptase to create a nick at the second position and use the second RTT as a template to extend the second strand of the target DNA molecule to form a second new single strand complementary to the second RTT, wherein the first new single strand and the second new single strand can form a double-stranded region to displace the target DNA sequence from the target DNA molecule.

25. One or more polynucleotides according to claim 24, further comprising or encoding a Cas protein and a reverse transcriptase.

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