WO2023141504A2

WO2023141504A2 - Dcas9-integrase for targeted genome editing

Info

Publication number: WO2023141504A2
Application number: PCT/US2023/060898
Authority: WO
Inventors: Eli Tijerina LYONS; Charlane Joy CARDOS; Elena Natsumi KAYAYAN
Original assignee: Genomeminer Inc Formally Tupac Bio Inc
Current assignee: Genomeminer Inc Formally Tupac Bio Inc
Priority date: 2022-01-19
Filing date: 2023-01-19
Publication date: 2023-07-27
Anticipated expiration: 2024-07-19
Also published as: WO2023141504A3

Abstract

A fusion protein for gene editing, which combines the catalytically dead, mutant Cas9 protein with the HIV-integrase enzyme, using a short linker sequence was developed. The resulting fusion protein is called dCas9-Integrase. The dCas9 integrase can be encoded in a single plasmid to contain a sequence that expresses the dCas9-Integrase protein and a crRNA sequence that is complementary to a target sequence in a host genome, creating a genome editing platform to modify any DNA sequence in the host genome.

Description

DCAS9-INTEGRASE FOR TARGETED GENOME EDITING

Cross References to Related Applications.

This application claims the benefit of Applicant’s prior provisional application, U.S. Ser. No. 63/300,652, filed on January 19, 2022.

Reference To Electronic Sequence Listing

The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on January 19, 2023 , is named “23-01-19_Sequences_ELI003PCT.xml” and is 89,918 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

Field of Invention.

The invention concerns an improved, targeted genome editing platform using the dCas9-Integrase fusion protein.

Background

Genome editing and gene therapy are of great interest for the prevention and treatment of diseases such as single gene disorder (e.g., sickle cell disease), cancer, heart disease, mental illness and HIV. See e.g., Park S.H., Lee C.M., et.al. Therapeutic Crisps/Cas9 Genome Editing for Treating Sickle Cell Disease. Blood 2016 128:4703, and complex diseases (e.g., fatal genetic diseases), England S.B., Nicholson L.V., Johnson M.A., Very Mild Muscular Dystrophy Associated with the Deletion of 46% of Dystrophin, Nature 1990; 343(6254). Genome editing has the ability to precisely and efficiently introduce a variety of genetic alterations into microbes, plant cells and mammalian cells via the insertion of a DNA sequence, the deletion of a DNA sequence or region, or the removal and replacement of one DNA sequence with another sequence. See e.g., Katare D.P. and Aeri V., Progress in Gene Therapy: A Review, I.J.T.P.R. 2010;l:33; Gardlik R., Palffy R., Hodosy J., Lukacs J., Turna J., Celec P., Vectors and Delivery Systems in Gene Therapy, Med Sci Monit. 2005; 11:110-21; and Wu Z., Yang H., Colosi P., Effect of Genome Size on AAV Vector Packaging, 2010 Mol. Ther. 18:80-86.

Genome editing facilitated by a CRISPR-Cas system complexes a protein and gRNA to guide and cleave specific DNA regions and incorporate foreign DNA into a precise location of a host genome. For example, some of these previous gene-editing techniques employ the CRISPR /Cas9 system. See e.g., Feng Zhang, Yan Wen, Xiong Guo, CRISPR/Cas9 for genome editing: progress, implications and challenges, Human Molecular Genetics, Volume 23, Issue Rl, 15 September 2014, Pages R40-R46, Liang Z, Zhang K, Chen K, Gao C. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J Genet Genomics. 2014 Feb 20;41(2):63-8. doi: 10.1016/j.jgg.2013.12.001. Epub 2013 Dec 14. PMID: 24576457, Epinat JC, Arnould S, Chames P, Rochaix P, Desfontaines D, Puzin C, Patin A, Zanghellini A, Piques F, Lacroix E. A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res. 2003 Jun 1 ;31 (11):2952-62. doi: 10.1093/nar/gkg375. PMID: 12771221 ; PMCID: PMC 156710, and artificially synthesized, fusion proteins (e.g., dCas9-Transposase, Target- AID) Strecker J, Ladha Am Gardner Z, Burgk S, Makarova K, Koonin E, Zhang F., RNA-guided DNA insertion with CRISPR-associated transposases. 2019 Jul 5;365(6448):48-53. doi: 10.1126/science. aax9181. Epub 2019 Jun 6 and Keiji Nishida, Takayuki Arazoe, Nozomu Yachie, Satomi Banno, Mika Kakimoto, Mayura Tabata, Masao Mochizuki, Aya Miyabe, Michihiro Araki, Kiyotaka Y. Hara, Zenpei Shimatani, Akihiko Kondo, Science 353,aaf8729 (2016), DOI: 10.1126/science.aaf8729. Integration using homology recombination and virus integration are common for insertion. However, there are limitations and/or shortcoming with these techniques.

Some of these previously developed gene-editing techniques are susceptible to off-target errors, can have low efficiency, can be immunogenic or toxic to target cells, are often expensive, time-consuming to use, difficult to engineer, and may have limited targetable sites. For example, these previously developed CRISPR-based techniques have been attributed to causing unwanted off target effects, which can alter the functions of the gene, which in turn can lead to genomic instability, that can potentially disrupt the normal function of genes, hindering CRISPR’s prospective application in clinical trials. See e.g., Goff S.P., Genetics of Retroviral Integration, Ann. Rev. Genet. 1992; 26:527-544; and Thyagarajan B., Olivares, E.C., et.al., Site-Specific Genomic Integration in Mammalian Cells Mediated by Phage C31 Integrase, Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP. Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol Cell Biol. 2001 Jun;21(12):3926-34. doi: 10.1128/MCB.21.12.3926-3934.2001. PMID: 11359900; PMCID: PMC87055.

Microbes are good targets for CRISPR-Cas system because they produce a variety of compounds ranging from fuels to chemicals and drugs. To improve the compound production yield from the laboratory set-up, bioreactors are used to scale up the compound synthesis. However, the culture conditions for commercially available bioreactors are often different from the laboratory flasks setting and it has been observed that some microbe strains do not grow equally in both environments. See e.g., Humphrey A., Shake Flask to Fermentor: What Have We Learned?, Biotechnol. Progress. 1998; 14: 3-7.

Plasmids are often used to express genes in a host microbial cell. These circular DNA pieces are the main vectors of horizontal gene transfer among bacteria for long term bacterial adaptation. Halary S., Leigh J.W., Cheaib B., Lopez P., Bapteste E., Network Analyses Structure Genetic Diversity in Independent Genetic Worlds, Proc. Natl Acad. Sci. USA 107, 127-132 (10.1073/pnas.0908978107 (2009)).

However, horizontal gene transfer can be a threat if resistance genes or other genes are transferred into endogenous microbial communities. Engineered microbes can reduce this problem by eliminating the use of plasmids and therefore the occurrence of horizontal gene transfer is minimized. Currently, few tools have been explored to perform genome editing and expression control of microbes. Among these are Zinc-finger nucleases (ZFN), antisense RNA approach, RNA interference (RNAi) and transcription activator-like effector nucleases (TALEN). However, using these approaches is time consuming and laborious compared to the CRISPR-Cas9 technology. CRISPR-Cas9 tools has been used to engineer model industrial organisms such as Saccharomyces cerevisiae and Escherichia coli to facilitate high product formation without fatal consequences to the host organism, therefore the integration of target gene into the genome is preferable. See e.g., Pingfang Tian, Jia Wang, Xiaolin Shen, Justin Forrest Rey, Qipeng Yuan, Yajun Yan, Fundamental CRISPR-Cas9 tools and current applications in microbial systems, Synthetic and Systems Biotechnology, Volume 2, Issue 3, 2017, Pages 219-225, ISSN 2405-805X, doi: https://doi.org/10.1101/423012; Zerbini. F., Zanella, I., Fraccascia, D. el al. Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli . Microh Cell Fact 16, 68 (2017). https://doi.org/10.1186/sl2934-017-0681-l; Esther Egger, Christopher Tauer, Monika Cserjan-Puschmann, Reingard Grabherr & Gerald Striedner, Fast and Antibiotic Free Genome Integration into Escherichia Coli Chromosome, Sci. Rep. 2020 Oct 5; 10(1): 16510. doi: 10.1038/s41598-020-73348-x. PMID: 33020519; PMCID: PMC7536200.

In summary, an improved gene editing tool is needed to reduce off-target effects, improve efficiency and increase the range of targetable sites in the host genome.

Summary

Disclosed herein is a novel and precise genome editing platform that can deliver a genetic element of interest to a precise location on a host genome with the one or more of the following benefits: low off target effects compared to similar gene-editing technologies available, ability to target any region in any genome with high precision; and low toxicity/susceptibility of causing an immune response.

A novel fusion protein, named dCas9-Integrase (or “dCas9-Int”), provides a customizable, genome-editing platform which improves upon previous genome-editing methods. Fusion proteins are formed by linking two proteins with a short linker sequence. dCas9-Int consists of the catalytically dead Cas9 protein linked to the HIV-integrase enzyme. The linker sequence may be SGSETPGTSESATPES (SEQ ID NO: 20), or some other sequence. The dCas9-Int may also include a start codon and/or a stop codon. The dCas9-Int fusion protein is then encoded into a backbone vector, resulting in a plasmid that expresses the catalytically dead mutant Cas9 (dCas9) linked to HIV Integrase by a short linker. The plasmid can contain a crRNA sequence that is complementary to the target sequence lacZ of the host genome HB101, however the sgRNA is customizable so that any crRNA can be cloned into the backbone dCas9-Int protein to target any DNA sequence in the host’s genome. dCas9-Int may also be encoded in a plasmid. For example, a plasmid may include a sequence that encodes a mutant Cas9 endonuclease from a Streptococcus pyogenes with a D10A mutation in the RuvC catalytic domain and a H480A mutation in the HNH catalytic domain and a sequence that encodes the HIV Integrase protein. The plasmid may also include a tracrRNA sequence and/or a crRNA sequence. The crRNA sequence in the plasmid may be customized to include a hybridization of a sequence to be modified in a host genome.

The plasmid may also include a sequence that encodes antibiotic resistance, such as a sequence that encodes Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin and/or Tetracycline resistance. The plasmid can further include a sequence that encodes an origin of replication, such as a pBR322 origin of replication and a Cas9 native promoter.

A plasmid containing a sequence that encodes the dCas9-Int fusion protein can be constructed with a sgRNA sequence and/or a sequence that encodes for antibiotic resistance. The sgRNA sequence may comprises a crRNA sequence and a tracrRNA sequence, where the crRNA sequence may be customizable to hybridize with a sequence on the host genome. The sequence that encodes for antibiotic resistance may be specific to Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin and/or Tetracycline. The dCas9-Int and sgRNA plasmid may include a sequence that encodes an origin of replication, such as a pBR322 origin of replication.

A pTransO4 plasmid may also be constructed with a sequence that includes U5*U3 long terminal repeats from an HIV-1 virus, a sequence that has multiple cloning sites, and a sequence that encodes antibiotic resistance genes. The pTransO4 plasmid may also include an origin of replication, such as the pl5A origin of replication, and/or a sequence that encodes antibiotic resistance genes. The antibiotic resistance can be resistance to one or more of the following antibiotics: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin and/or Tetracycline.

One or more of the previously mentioned plasmids can be introduced into a cell. For example, a cell can express a dCas9-Int plasmid and a pTransO4 plasmid. The dCas9-Int plasmid may or may not include an origin of replication such as the pBR322 origin of replication, and the pTransO4 plasmid may or may not include a different origin of replication, such as the pl5A origin of replication. In addition, the dCas9-Int plasmid might include a tracrRNA sequence and/or a crRNA sequence. The crRNA sequence may be customizable to include a hybridization of a sequence to be modified in a host genome. The dCas9-Int plasmid may include a sequence that encodes antibiotic resistance, such as a sequence that encodes resistance to one or more of the following antibiotics: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin or Tetracycline.

One of more of the previously mentioned plasmids or fusion proteins can be used to modify a target sequence in a host genome. For example, a dCas9-Int plasmid can include a customizable crRNA sequence that is capable of hybridizing to the target sequence in the host genome. The dCas9-Int plasmid can be introduced into a host cell, where the customizable crRNA sequence hybridizes to the target sequence in the host genome, causing the target sequence in the host genome to be modified. The host cell can be a eukaryotic cell, a plant cell, an algae cell, a non-human cell, or a mammalian cell. The dCas9-Int plasmid may include a sequence that encodes for antibiotic resistance, including one or more of the following antibiotics: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin or Tetracycline. Further the dCas9-Int plasmid may include an origin of replication, and the customizable crRNA sequence may be linked to a tracrRNA sequence. dCas9-Int can also be combined with sgRNA to form a complex. The complex can be used with a plasmid which contains a genetic element of interest to modify a host genome. The genetic element of interest may include a sequence that can hybridize with a target sequence in the host genome. The plasmid may include a sequence that encodes for antibiotic resistance, such as one or more of the following antibiotics: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin or Tetracycline. The plasmid may also include an origin of replication. The sgRNA protein may be comprised of crRNA and tracrRNA.

Brief Description of the Drawings

Some of the novel features of this disclosure are best understood from the accompanying drawings, viewed in conjunction with the accompanying description.

Fig. 1(a) illustrates a dCas9-Integrase fusion protein.

Fig. 1(b) (SEQ ID NO: 1) illustrates a sequence for the dCas9-Integrase fusion protein.

Fig. 1(c) illustrates the dCas9-Integrase targeting mechanism.

Fig. 2(a) illustrates an sgRNA sequence, a dCas9-Integrase protein and a linear genetic element of interest.

Fig. 2(b) illustrates a complex formed from the dCas9-Integrase, sgRNA, a linear genetic element of interest and a target sequence on the host genome.

Fig. 2(c) illustrates a target modification to the host genome caused by the complex shown in Fig. 2(b).

Fig. 2(d) illustrates the components of a complex to be formed, specifically, dCas9-Integrase, sgRNA, and a circular genetic element of interest.

Fig. 2(e) illustrates a complex formed from dCas9-Integrase, sgRNA, a circular genetic element of interest and a target on the host genome. Fig. 2(f) illustrates a resulting target modification to the host genome, where the target sequence on the host genome is replaced with a sequence from the circular genetic element of interest.

Fig. 3(a) (SEQ ID NO: 2) illustrates a sequence of the pFusion02.1 plasmid.

Fig. 3(b) illustrates an embodiment of the pFusion02.2 plasmid.

Fig. 3(c) (SEQ ID NO: 3) illustrates a sequence for the pFusion02.2 plasmid.

Fig. 4(a) illustrates an embodiment of the pFusion02.3 plasmid.

Fig. 4(b) (SEQ ID NO: 4) illustrates a sequence for the pFusion02.3 plasmid.

Fig. 5(a) illustrates an embodiment of the pFusion02.4 plasmid.

Fig. 5(b) illustrates crRNA fused to tracrRNA to form an sgRNA molecule for the pFusion02.4 plasmid.

Fig. 5(c) (SEQ ID NO: 5) illustrates a sequence for the pFusion02.4 plasmid.

Fig. 6 (SEQ ID NO: 6) illustrates a sequence for the pFusion02.5 plasmid.

Fig. 7(a) illustrates an embodiment of the pFusion03(mammalian) plasmid.

Fig. 7(b) illustrates an alternative embodiment of the pFusion03(mammalian) plasmid.

Fig. 7(c) illustrates crRNA fused to tracrRNA to form an sgRNA molecule for the pFusion03 (mammalian) plasmid.

Fig. 7(d) (SEQ ID NO: 7) illustrates a sequence for the pFusion03(mammalian) plasmid.

Fig. 8 (SEQ ID NO: 8) illustrates a sequence for the pSP72B vector. Fig. 9(a) illustrates an embodiment of the pTransO4 plasmid.

Fig. 9(b) illustrates an embodiment of the pTransO4 plasmid with the origin of replication removed.

Fig. 9(c) (SEQ ID NO: 9) illustrates a sequence for the pTransO4 plasmid.

Fig. 10(a) illustrates a custom-designed guiding crRNA sequence.

Fig. 10(b) illustrates a tracrRNA sequence.

Fig. 11 illustrates an embodiment where the pFusion02.2 plasmid and the pTransO4 plasmid are both expressed in the same cell.

Fig. 12 (SEQ ID NO: 10) illustrates a pBR322 origin of replication sequence.

Detailed Description

Definitions genetic element: A DNA sequence that is integrated into any gene of a target genome. dCas9: A catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system. It is an RNA-guided, DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain. dCas9 is used in CRISPR systems along with gRNAs to target specific host genome locations.

Integrase: An enzyme produced by a retrovirus (e.g., HIV) that integrates its DNA into a host genome's DNA. linker: A short sequence of DNA that connects the dCas9 protein with Integrase. single-guide RNA (sgRNA): An RNA sequence that contains both a custom-designed crRNA sequence fused to a scaffold tracrRNA sequence. fusion protein system: A system that consists of dCas9 and the Integrase protein joined together by a linker. plasmid: A circular, double-stranded DNA molecule that is physically separated from chromosomal DNA and replicates itself independently from chromosomal DNA. transformation: The process of horizontal gene transfer wherein a foreign DNA strand is taken up by bacterial cells. transfection: The process of intaking foreign DNA or purified nucleic acid into eukaryotic cells. off-target error: An unintended genetic modification in the host genome resulting from the application of gene editing techniques.

Chemical DNA synthesis: The process of synthesizing DNA sequences using chemical methods which are usually carried out when the DNA sequence does not occur naturally or contains base modifications.

DNA Sequence: A sequence consisting of DNA nucleotides.

Hybridizing: combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double- stranded molecule through base pairing.

Protein Sequence: A sequence consisting of amino acids. host genome: A genome carried by a host organism, whether a prokaryote or eukaryote, to be edited. gene editing or editing: inserting, removing, or removing then replacing one or more nucleotides in a host’s genome.

The following is a detailed description to illustrate the principles and various aspects of the different embodiments of the disclosure. The disclosed embodiments are illustrative, not restrictive. The scope of the disclosure, as shown in the following embodiments, encompasses numerous alternatives, modifications and equivalents, and is limited only by the claims, not by this description. In addition, for the purpose of clarity, some of the basic aspects of molecular genetics, CRISPR and gene editing have not been described so that various aspects of the embodiments are not unnecessarily obscured. Referring to Fig. 1(a), the dCas9-Int fusion protein is shown. As shown in Fig. 1(a), the structure of the dCas-Int protein includes a start codon, followed by a catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system. dCas9 is an RNA-guided, DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and H840A mutation in the HNH catalytic domain. The dCas9 protein is followed by a short linker that links the dCas9 to the HIV Integrase protein. The most efficient linker was used in the fusion protein described in Guillinger 2014. Fokl-L8, specially: SGSETPGTSESATPES (SEQ ID NO: 20). Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification, Nat Biotechnol. 2014 Jun;32(6):577-582. doi: 10.1038/nbt.2909. Epub 2014 Apr 25. PM ID: 24770324; PMCID: PMC4263420. The HIV Integrase gene is inserted after the linker, such that the HIV Integrase sequence is from GenBank: L21188.1. Finally, the dCas9-Int protein is terminated with stop codon, as shown in Fig. 1(a). The HIV Integrase protein allows efficient integration of DNA into a target DNA sequence. The sequence for dCas9-Int protein is shown Fig. 1(b) and consists of 1367 amino acids (SEQ ID NO: 1). dCas9-Int provides a customizable genome-editing platform which improves upon previous genome-editing methods by linking two proteins, resulting in the creation of a novel fusion protein, dCas9-Int. dCas9-Int can be encoded into backbone vector, resulting in a plasmid that expresses the fusion protein of catalytically dead-Cas9 (dCas9) linked to HIV integrase by a short linker.

The dCas9-Integrase fusion protein can be assembled as follows. The pSP72A vector is digested at Hpal and Aatll restriction sites to clone the insert dCas9-Integrase sequence. For the insert fusion protein, start from the sequences [‘upstream of tracrRNA' until ‘after Direct repeat (DR)'] from the Bhatt et. al 2019 (infra) plasmid sequence pRC2311 (with pl promoter from Michael Tellier, Ronald Chalmers, A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition BioRxiv 2019, doi: https://doi.org/10.1101/423012. Shiva m Bhatt, Ronald Chalmers, Targeted DNA transposition in vitro using a dCas9-transposase fusion protein, Nucleic Acids Research, Volume 47, Issue 15, 05 September 2019, Pages 8126-8135. Remove the transposase gene and the stop codon after dCas9. Elements of the plasmid were identified through alignment of the pdCas9 plasmid (see https:/ www.addgene.org/46569/) and the pRC2311. The promoter, dCas9 gene, and other plasmid elements are also verified. As previously explained, the linker was identified by Guillinger (2014)(supra). The HIV Integrase gene is inserted after the linker and the sequence is from GenBank: L21188.1

Other methods of creating the physical embodiment of the dCas9-Integrase fusion protein include:

1. Artificial protein mimics (Example of artificial ‘protein mimics' being produced by ‘forcing' peptides together =

<https:/ www .sciencedaily.com/releases/2016/08/ 160809143608 ,htm>) ;

2. Self-assembly (Example of self-assembling protein =

<https:/ www .sciencedirect.com/science/article/pii/S20Q 103701530009X>) ; and

3. Protein translation from synthesized mRNA (Example of mRNA being directly artificially synthesized =

<https:/ www.ncbi.nlm.nih.gov/pmc/articles/PMC2995060/>).

Fig. 1(c) illustrates an embodiment that demonstrates the dCas9-Int targeting and editing mechanisms in operation. First, a cell is transfected with a plasmid that expresses the dCas9-Int protein and an appropriately designed sgRNA sequence. The sgRNA is comprised of a crRNA sequence, GTCACGACGTTGTAAAACGA (SEQ ID NO: 12), linked to scaffold tracrRNA sequence. The crRNA sequence is complimentary to the target sequence CAGTGCTGCAACATTTTGCT (SEQ ID NO: 13) in the host genome. The crRNA recognizes the target DNA sequence in the host’s genome, just upstream of the PAM sequence (in this case GCC) and binds to that portion of the host’s genome. The dCas9-Int then cuts both strands of DNA to match the crRNA sequence, allowing a replacement DNA sequence to be inserted into a precise location in the host’s genome. dCas9-Int can be combined with other components to form a gene editing mechanism. For example, as shown in Fig. 2(a), dCas9-Int can be combined with sgRNA and linear genetic element of interest to form a complex. Although the use of dCas9-Int is mentioned in W02016/161207 in conjunction with a linear genetic element of interest, the inventors herein have determined experimentally that a linear genetic element of interest is not suitable if dCas9-Int is to be used for gene editing. According to our experiments, a linear genetic element of interest is easily digested by nucleases inside the cell, whereas circular genetic elements of interest minimize the degradation and are able to replicate. Accordingly, a circular genetic element of interest is preferrable, as shown in Figs. 2(d) and 2(e) (discussed further below). dCas9-Int, sgRNA and a circular genetic element of interest can be combined to form a complex that can modify a target sequence in the host’s genome, as shown in Fig. 2(b). As shown in Fig. 2(c), the target sequence in the host genome is replaced with the genetic element of interest.

Fig. 2(d) shows the components of a gene editing mechanism, including a circular genetic element of interest (i.e., a plasmid), a fusion protein such as dCas9-Int, and sgRNA. Fig. 2(e) shows a complex formation consisting of dCas9-Int, sgRNA, and a circular genetic element of interest annealed to a target sequence in the host genome. Fig. 2(f) shows the genetic element of interest that has replaced the target sequence in the host genome. As shown in Fig. 3(b), dCas9-Int can be incorporated into a plasmid, such as the pFusion02.2 plasmid. Of course, dCas9-Int can be incorporated into a variety of other plasmids as well. One way to construct these plasmids is through DNA synthesis. The following elements are included in the pFusion02.1 and pFusion02.2 plasmids: dCas9-Int, which is the fusion of a catalytically dead Cas9 protein linked to the Integrase protein by a short linker, and an sgRNA sequence that contains customizable crRNA and tracrRNA, where the crRNA binds to the target sequence in the host’s genome. Some optional elements include: (1) ampR: a sequence that codes for the ampicillin resistant gene for antibiotic selection (genes resistant to other antibiotics can be used interchangeably); (2) a stuffer sequence: a short DNA sequence (e.g., GAGACGAGTCTCGGAAGCTCAAACGTCTC)(SEQ ID NO: 14), that can be digested by the BsmBI restriction enzyme and cloned with any unique crRNA sequences; and (3) ORI: a low-copy Origin of Replication, such as pBR322, to initiate the plasmid replication. For the pFusion02.2 plasmid depicted in Fig. 3(b), the tracrRNA is controlled by a downstream promotor while the dCas9-Int is controlled by an upstream, Cas9 native promoter.

The sequence for the pFusion02.1 plasmid (SEQ ID NO: 2) is shown in Figure 3(a) and the sequence for the pFusion02.2 plasmid (SEQ ID NO: 3) is shown in Figure 3(c). As shown in Fig. 3(a), one embodiment of the pFusion02.1 plasmid consists of 8163 base pairs, including a sequence that codes for Ampicillin resistance (a sequence that codes for a different type of antibiotic resistance can also be used); a sequence that codes for a tracrRNA; and a sequence that codes for the dCas9-Int protein. As shown in Fig. 3(c), one embodiment of the pFusion02.2 plasmid consists of 8204 base pairs, including a sequence that codes for the pBR322 origin of replication; a sequence that codes for Ampicillin resistance (a sequence that codes for a different type of antibiotic resistance can also be used); a sequence that codes for a tracrRNA and a sequence that codes for the dCas9-Int protein. For the crRNA sequence, short oligonucleotides (oligos) a and b are synthesized separately and annealed. The pSP72A plasmid is digested with the BsmBI restriction enzyme and cloned with the annealed oligos. The oligo sequences are as follows:

Oligo a: AAACGGTTTTCCCAGTCACGACGTTGTAAAACGAG (SEQ ID NO:

15)

Oligo b: AAAACTCGTTTTACAACGTCGTGACTGGGAAAACC (SEQ ID NO:

16)

When a and b are annealed, the following annealed fragment is formed:

AAACGGTTTTCCCAGTCACGACGTTGTAAAACGAG (SEQ ID NO: 17)

CCAAAAGGGTCAGTGCTGCAACATTTTGCTCAAAA (SEQ ID NO: 18)

See Supplementary Table 2 of Shivam Bhatt, Ronald Chalmers, Targeted DNA transposition in vitro using a dCas9-transposase fusion protein, Nucleic Acids Research, Volume 47, Issue 15, 05 September 2019, Pages 8126-8135, for further details.

Fig. 4(a) shows another embodiment, the pFusion02.3 plasmid, which is a dCas9-Int along with a pFusion02.2 plasmid that had its origin of replication (“ORI”) removed using digestion enzymes and was self-ligated to re-circulize the plasmid. Removal of the ORI prevents the plasmid from replicating after transformation. Low expression and transient expression of the fusion protein avoids problems that can result from too many copies of the fusion protein plasmids. The sequence for the pFusion02.3 plasmid (SEQ ID NO: 4) is illustrated in Fig. 4(b).

Fig. 5(a) is an embodiment of the pFusion02.4 plasmid. As show in Fig. 5(a), the pFusion02.4 plasmid comprises a sequence that expresses the dCas9-Int fusion protein, a sequence that expresses sgRNA, a sequence that expresses Ampicillin resistance gene and an ORI. The sequences that express other types of antibiotic resistances can be substituted for the sequence that expresses Ampicillin resistance. Also as shown in Fig. 5(a), dCas9-Int and sgRNA sequences are controlled by upstream promotors, while the sequence that expresses Ampicillin resistance is controlled by a downstream promoter. Fig. 5(b) illustrates sgRNA for the pFusion02.4 plasmid.

Fig. 5(c) illustrates an embodiment of the pFusion02.4 plasmid sequence (SEQ ID NO: 5). As shown in Fig. 5(c), the pFusion02.4 plasmid consists of 8204 base pairs, including a sequence that codes for the pBR322 origin of replication, a sequence that codes for Ampicillin (or other types of antibiotic) resistance, a sequence that codes for a tracrRNA sequence and a sequence that codes for the dCas-Int protein.

Fig. 6 illustrates an embodiment of the pFusion02.5 plasmid sequence. According to Fig. 6, one embodiment (SEQ ID NO: 6) of the pFusion02.5 plasmid consists of 7576 base pairs, including a sequence that codes for Ampicillin (or some other type of antibiotic) resistance, a sequence that codes for a tracrRNA sequence and a sequence that codes for the dCas-Int protein.

Fig. 7(a) illustrates an embodiment of the pFusion03 plasmid for mammalian cell expression. The pFusion03 plasmid shown in Fig. 7(a) includes a sequence that codes for the dCas9-Int fusion protein; an ORI; a sequence that codes for Ampicillin resistance (or other types of antibiotic resistance can be substituted for Ampicillin resistance); a sequence that codes for sgRNA that targets the chr4:58110375-58110396 region in the host genome, (or other sequences that codes for sgRNA that targets any region in the host genome); and three promoters that regulate the sgRNA, dCas9-Int and ampR elements. The sgRNA and dCas-Int elements are designed to create a complex that allows a custom sequence of the host’s genome to be modified.

Fig. 7(b) illustrates an alternative embodiment of the pFusion03 (mammalian) plasmid. As shown in Fig. 7(b), the pFusion03 plasmid includes a sequence that expresses the dCas9-Int fusion protein, a sequence that expresses a customizable sgRNA that targets a portion of the host genome, and a sequence that expresses Ampicillin resistance (sequences that express other types of antibiotic resistance can also be used). Also as shown in Fig. 7(b), the sequences that code for dCas9-Int and sgRNA are each controlled by a separate upstream promoter. The sequence that codes for the Ampicillin resistance is controlled by a downstream promoter. Fig. 7(c) illustrates crRNA fused to tracrRNA to form an sgRNA molecule for the pFusion03 (mammalian) plasmid. Fig. 7(d) illustrates a sequence for the pFusion03 (mammalian) plasmid (SEQ ID NO: 7). A related embodiment sequence is pFusion03 mammalian-base plasmid sequence (SEQ ID NO: 11).

Fig. 8 illustrates a sequence (SEQ ID NO: 8) for the pSP72B vector, which is used to construct a mammalian plasmid. The pSP72B vector is made by modifying a pSP72 plasmid from Promega, digested at Aatll restriction site to insert the multi-cloning Sequence A, to obtain the pSP72A vector. The pSP72A is then digested at the Ndel and Hpal restriction sites to insert a sequence with additional restriction sites (Sequence of Notl insert linear fragment = CATATGCGGCCGCGTT)(SEQ ID NO: 19) (Sequence B). This adds additional restriction site Notl (while conserving Ndel and Hpal recognition sites) for the insertion of Sequence B, for the purpose of mammalian plasmid construction. In this case, the Multiple Cloning Site (MCS) 1 is removed, while all of MCS 2 is conserved.

Figs. 9(a) and 9(b) illustrate an embodiment a pTransO4 plasmid, which can be constructed as follows with the necessary elements to allow efficient ligation into the target genome. The pTrans-04 is a small circular, low-copy or no-copy plasmid containing the

U5*U3 LTRs region and optional Kanamycin resistant (or other antibiotic resistant) genes for antibiotic selection. Some of the key elements of the pTransO4 transgene plasmid are as follows:

1. a low copy ORI such as the pl5A origin of replication (pl5A ORI).

<http s :/ w w w . addgene . org/search/c atalog/plasmids/?q=p 15 A+ori+lo w+copy> . (Note :

<https:/ www.addgene.org/74088/> for the sequence source.

2. an ORI in the transgene plasmid and the fusion Plasmid such that they may coexist inside a bacterial cell;

3. a spacer sequence: a lOObp upstream and downstream of the ORI;

4. the U5*U3 LTRs, which were designed by Ritchetta et. al Clemence Richetta, Sylvain Thierry, Eloise Thierry, et.al Two-long terminal repeat (LTR) DNA circles are a substrate for HIV-1 integrase, Journal of Biological Chemistry, 2019, DOI:https://doi.org/10.1074/jbc.RAl 18.006755 ];

5. a Multi-Cloning Site (MCS) used for inserting additional DNA sequences into the plasmid using a molecular cloning; and

6. an antibiotic resistant gene, such as a gene that is resistant to kanamycin (genes that are resistant to other antibiotics can be used interchangeably).

Fig. 9(b) shows the pTransO4 (no ORI) plasmid, specifically, the origin of replication was removed using digestion enzymes and was self-ligated to re-circulize the plasmid. Note that the antibiotic resistance gene is optional. Of course, for the pTransO4 (no ORI) plasmid, replication does not occur after transformation. Fig. 9(c) illustrates a sequence for the pTransO4 plasmid (SEQ ID NO: 9).

Figs. 10(a) and 10(b) illustrates sgRNA, composed of a crRNA sequence linked to tracrRNA. Starting from the 5’ end, Fig. 10(a) shows a guiding sequence composed of crRNA. The crRNA includes a custom-designed targeting sequence that is complimentary to the target DNA sequence in the host’s genome. For example, the crRNA sequence is 20 plus base pairs, and because it is complimentary to the DNA of interest in the host’s genome, it directs the dCas9 nuclease activity to that region on the host genome. Fig. 10(b) illustrates tracrRNA, fused to the crRNA. The crRNA hybridizes with the tracrRNA to form sgRNA.

Fig. 11 illustrates an embodiment where a bacterial cell expresses both the pFusion02.2 plasmid and the pTRANS-04 plasmid. Both plasmids will be able to co-exist inside a single cell by designing each plasmid to have different ORI (e.g., one plasmid can have a pBR322 ORI, while the other plasmid can have a pl5A ORI), or by synthesizing one of the plasmids so that it does not contain an ORI. Specifically, the 2 plasmids can co-exist in a single cell if the respective origins of replication are different or only 1 plasmid has an origin of replication. For example, the pFusion02.2 plasmid can express the dCas9-Int fusion protein, while pTRANS-04 plasmid can express multiple cloning sites in order to clone different sgRNAs for specific target sequences.

Fig. 12 is an embodiment (SEQ ID NO: 10) of a pBR322 origin of replication sequence. As shown in Fig. 12, the pBR322 origin of replication sequence consists of 648 base pairs from the pBR322 plasmid, created in 1977 in the laboratory of Herbert Boyer at the University of California. The 648 base pairs of pBR322 shown in Fig. 12 are well-suited as an origin of replication for the pFusion02.2 and pFusion02.3 plasmids, disclosed herein, although there are other sequences that can be used for the origin of replication for these plasmids.

The fusion proteins described herein can also be produced independently and used in combination with synthetic sgRNA. Also, by way of example, the fusion proteins described herein can be used without transformation or transfection of DNA plasmids coding for such a fusion protein and related elements such as sgRNA. Further, the plasmids disclosed herein can be synthesized or assembled using molecular cloning techniques, or copied through transformation and amplification in e.coli or other microbes, as these techniques are well known.

Various embodiments can be obtained by means of chemical DNA synthesis, or could be made using molecular biology cloning and DNA construction techniques to obtain a plasmid expressing the dCas9-Int fusion protein. A plasmid that expresses a dCas9-Int protein could then be applied in combination with various molecular biology techniques such as transformation and transfection to edit any target DNA sequence in many types of organisms. Certain aspects of the gene editing system described herein can be purchased and modified. For example, crRNA can be synthesized and inserted into the backbone plasmid in order for genome edits to occur at a custom target of interest. With this, genome editing can occur at any target location in the host genome.

Claims

What is Claimed is:

1. A composition comprising: [e.g., dCas9-Int] a mutant Cas9 endonuclease from a Streptococcus pyogenes, with a D10A mutation in the RuvC catalytic domain and a H480A mutation in the HNH catalytic domain; an HIV Integrase protein; and a linking sequence that links the mutant Cas9 endonuclease to the HIV Integrase.

2. The composition of claim 1 further comprising start codon and stop codon.

3. The composition of claim 1 wherein the linking sequence is SGSETPGTSESATPES (SEQ ID NO; 20).

4. The composition of any one of claims 2-3 wherein the linking sequence is SGSETPGTSESATPES (SEQ ID NO: 20).

5. A plasmid comprising: [e.g., a generic plasmid that encodes dCas9-Int] a sequence that encodes a mutant Cas9 endonuclease from a Streptococcus pyogenes with a D10A mutation in the RuvC catalytic domain and a H480A mutation in the HNH catalytic domain; and a sequence that encodes the HIV Integrase protein.

6. The plasmid of claim 5 further comprising a tracrRNA sequence.

7. The plasmid of claim 6 further comprising a crRNA sequence.

8. The plasmid of claim 7 wherein the crRNA sequence is customizable to include a hybridization of a sequence to be modified in a host genome. 9. The plasmid of claim 5 further comprising a sequence that encodes antibiotic resistance. 10. The plasmid of claim 5 wherein the sequence that encodes antibiotic resistance is specific to one or more of the following antibiotics: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin and/or Tetracycline. 11. The plasmid of claim 5 further comprising a sequence that encodes an origin of replication. 12. The plasmid of claim 5 further comprising a sequence that encodes for a pBR322 origin of replication. 13. A plasmid comprising [e.g., pFusion02.1 and pFusion02.3] a sequence that encodes a mutant Cas9 endonuclease from a Streptococcus pyogenes with a D10A mutation in the RuvC catalytic domain and a H480A mutation in the HNH catalytic domain; a sequence that encodes the HIV Integrase protein; a crRNA sequence; a sequence that encodes for antibiotic resistance; and a tracrRNA sequence. The plasmid of claim 13 further comprising a sequence that encodes an origin of replication. The plasmid of claim 13 further comprising [e.g., pFusion02.2] a sequence that encodes for a pBR322 origin of replication. The plasmid of 13 wherein the antibiotic resistance is resistant to Ampicillin. The plasmid of claim 13 wherein the sequence that encodes antibiotic resistance is specific to one or more of the following antibiotics: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin and/or Tetracycline. The plasmid of claim 13 wherein the crRNA sequence is customizable to hybridize with a sequence on the host genome. The plasmid of claim 13 further comprising a Cas9 native promoter. A plasmid comprising [e.g., pFusion02.5] a sequence that encodes a mutant Cas9 endonuclease from a Streptococcus pyogenes with a D10A mutation in the RuvC catalytic domain and a H480A mutation in the HNH catalytic domain; a sequence that encodes the HIV Integrase protein; an sgRNA sequence; and a sequence that encodes for antibiotic resistance.

21. The plasmid of claim 20 wherein the sgRNA comprises a crRNA sequence and a tracrRNA sequence. 22. The plasmid of claim 21 wherein the crRNA sequence is customizable to hybridize with a sequence on the host genome. 23. The plasmid of claim 20 wherein the antibiotic resistance is resistant to Ampicillin. 24. The plasmid of claim 20 wherein the sequence that encodes antibiotic resistance is specific to one or more of the following antibiotics: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin and/or Tetracycline. 25. The plasmid of claim 20 or claim 21 further comprising [e.g., pFusion02.4] a sequence that encodes an origin of replication. 26. The plasmid of claim 20 or 21further comprising a sequence that encodes a pBR322 origin of replication. 27. A plasmid comprising [e.g., pFusion03] a sequence that encodes a mutant Cas9 endonuclease from a Streptococcus pyogenes with a D10A mutation in the RuvC catalytic domain and a H480A mutation in the HNH catalytic domain; a sequence that encodes a HIV Integrase protein; a sequence that encodes an origin of replication; a sequence that encodes antibiotic resistance; and an sgRNA sequence.

28. The plasmid of claim 27 wherein the origin of replication is a pBR322 origin of replication. 29. The plasmid of claim 27 wherein the sgRNA comprises a crRNA sequence and a tracrRNA sequence. 30. The plasmid of claim 29 wherein the crRNA sequence is customizable to hybridize with a sequence on the host genome. 31. The plasmid of claim 27 wherein the antibiotic resistance is resistant to Ampicillin. 32. The plasmid of claim 27 wherein the sequence that encodes antibiotic resistance is specific to one or more of the following antibiotics: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin and/or Tetracycline. 33. A plasmid comprising [e.g., pTrans04 - no ori] a sequence that includes U5*U3 long terminal repeats from an HIV-1 virus; and a sequence that includes multiple cloning sites; and a sequence that encodes antibiotic resistance. 34. The plasmid of claim 33 further comprising an origin of replication. [e.g., pTrans04 with ori] 35. The plasmid of claim 33 further comprising a p15A origin of replication.

36. The plasmid of claim 34 further comprising where the antibiotic resistance is resistant to kanamycin. 37. The plasmid of claim 35 wherein the antibiotic resistance is resistant to one or more of the following antibiotics: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin and/or Tetracycline. 38. A cell that expresses 2 or more plasmids comprising [e.g., 2 plasmid embodiment, e.g. pFusion02.2 and pTrans04]. a first plasmid comprising, a sequence that encodes a mutant Cas9 endonuclease from a Streptococcus pyogenes with a D10A mutation in a RuvC catalytic domain and a H480A mutation in a HNH catalytic domain; a sequence that encodes a HIV Integrase protein; and a second plasmid comprising, a sequence that includes U5*U3 long terminal repeats from an HIV-1 virus; a sequence that includes multiple cloning sites; a sequence that encodes antibiotic resistance; and a sequence that includes an origin of replication. 39. The cell of claim 38 wherein the origin of replication for the second plasmid is a p15A origin of replication. 40. The cell of claim 38 wherein the first plasmid further comprises an origin of replication.

41. The cell of claim 38 wherein the first plasmid further comprises a pBR322 origin of replication. 42. The cell of claim 38 wherein the first plasmid further comprises a tracrRNA sequence. 43. The cell of claim 38 wherein the first plasmid further comprises a crRNA sequence. 44. The cell of claim 38 wherein the first plasmid further comprises a crRNA sequence that is customizable to include a hybridization of a sequence to be modified in a host genome. 45. The cell of claim 38 wherein the first plasmid further comprises a sequence that encodes antibiotic resistance. 46. The cell of claim 38 wherein the first plasmid further comprises a sequence that encodes Ampicillin resistance. 47. The cell of claim 39 wherein the first plasmid further comprises a sequence that encodes one or more of the following types of antibiotic resistance: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin or Tetracycline. 48. The cell of claim 38 wherein the sequence the encodes for antibiotic resistance in the second plasmid includes one or more of the following types of antibiotic resistance: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin and/or Tetracycline.

49. A method of modifying a target sequence in a host genome comprising, [e.g., gene editing using dCas9-Int] introducing a plasmid into a host cell, the plasmid comprising a sequence that encodes a mutant Cas9 endonuclease from a Streptococcus pyogenes with a D10A mutation in a RuvC catalytic domain and a H480A mutation in a HNH catalytic domain; a sequence that encodes a HIV Integrase protein; and a customizable crRNA sequence that is capable of hybridizing to the target sequence in the host genome, wherein the customizable crRNA sequence hybridizes to the target sequence in the host genome; and causing the target sequence in the host genome to be modified. 50. The method of claim 49 wherein the host cell is a eukaryotic cell. 51. The method of claim 49 wherein the plasmid further comprises an origin of replication. 52. The method of claim 49 wherein the plasmid further comprises a sequence that encodes antibiotic resistance. 53. The method of claim 49 wherein the plasmid further comprises a sequence that encodes one or more of the following types of antibiotic resistance: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin or Tetracycline. 54. The method of claim 49 wherein the customizable crRNA sequence is linked to a tracrRNA sequence.

55. The method of claim 49 wherein the host cell is from a non-human organism. 56. The method of claim 49 wherein the host cell is from a plant or algae. 57. The method of claim 49 wherein the host cell is a mammalian cell. 58. A complex comprising [e.g., dCas9-Int + sgRNA complex – Figs. 2d, e and f] a sequence that encodes a mutant Cas9 endonuclease from a Streptococcus pyogenes with a D10A mutation in a RuvC catalytic domain and a H480A mutation in a HNH catalytic domain linked to a sequence that encodes a HIV Integrase protein; an sgRNA protein, and a plasmid containing a genetic element of interest. 59. The complex of claim 58 wherein a sequence in the genetic element of interest is capable of hybridizing with a target sequence in host genome. 60. The complex of claim 58 wherein the plasmid includes a sequence that encodes for antibiotic resistance. 61. The complex of claim 58 wherein the plasmid further comprises a sequence that encodes one or more of the following types of antibiotic resistance: Ampicillin, Carbenicillin, Chloramphenicol, Hygromycin B, Kanamycin, Spectinomycin or Tetracycline. 62. The complex of claim 58 wherein the plasmid includes an origin of replication.

63. The complex of claim 58 wherein the sgRNA protein is comprised of crRNA and tracrRNA.