CN117264998A - Dual-function genome editing system and use thereof - Google Patents
Dual-function genome editing system and use thereof Download PDFInfo
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- CN117264998A CN117264998A CN202311193500.7A CN202311193500A CN117264998A CN 117264998 A CN117264998 A CN 117264998A CN 202311193500 A CN202311193500 A CN 202311193500A CN 117264998 A CN117264998 A CN 117264998A
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Abstract
The present invention relates to the field of genetic engineering. In particular, the present invention relates to genome editing fusion proteins comprising a CRISPR effector protein domain and a deaminase domain, as well as to bifunctional genome editing systems comprising said genome editing fusion proteins and uses thereof.
Description
The application is a divisional application of Chinese invention patent application with the application number of 202010660449.6, the application date of 2020, 7 months and 10 days and the invention name of 'dual-function genome editing system and application'.
Technical Field
The present invention relates to the field of genetic engineering. In particular, the present invention relates to genome editing fusion proteins comprising a CRISPR nuclease domain and a deaminase domain, as well as to bifunctional genome editing systems comprising said genome fusion proteins and uses thereof.
Background
The CRISPR/Cas9 gene editing system is characterized in that Cas9 binds and cleaves Double-stranded DNA of a target gene under the guidance of guide RNA to form Double-strand break (DSB), and insertion and/or deletion with different fragment lengths are introduced in the repairing process of an organism. The system is simple, convenient and efficient, and is widely applied to research and utilization of gene functions. However, it produces an editing effect with uncertainty, and it is difficult to realize site-directed editing of genes. Whereas the single base editing system formed by the fusion of nCas9 (D10A) with cytosine deaminase (Cytosine deaminase) achieves cytosine to thymine conversion without double strand breaks, efficient site-directed mutagenesis has been performed in plant, animal and human cells. These two gene editing systems have been widely used in disease treatment studies, animal model building, plant genetic breeding. However, these two gene editing systems have different working principles, different application ranges, and the editing results are not completely the same.
In genetic improvement of plants or even biomedical research, it is often necessary to edit multiple genes or different regions of one gene simultaneously, resulting in knockouts (indels) of specific gene fragments and substitutions of specific nucleotides. There are two strategies to achieve this result according to existing gene editing tools. One is a co-transformation system for knockout and base substitution (sgRNA, spCas9 and nCas 9-apodec 1 or A3A-PBE), which requires a large amount of exogenous DNA for a single transformation, which is a challenge to the transformation process, while a large amount of exogenous DNA is highly toxic to cells. In addition, the efficiency of obtaining the target plant containing both the base substitution and the indel mutation is low, and the screening workload is huge. The other method is batch conversion, firstly converting sgRNA and Cas9 for gene knockout, obtaining mutant plants containing target gene indel by tissue culture screening, and then carrying out secondary conversion by using the mutant plants containing target gene indel as receptor materials by a single base editing system.
To date, there is no gene editing system that can achieve both efficient base substitution and indel. Efficient C-to-T substitution and indel at specific locations for multiple species including plants are achieved simultaneously by a single transformation experiment using a dual function system. Guide RNAs of different lengths control the editing of target genomic loci, producing diverse mutations in coding and non-coding regions. Especially for some crop varieties which are difficult to transform or have long transformation period, the period can be greatly shortened, and the breeding process can be accelerated; the dual-function system can also be used for researching the action relation between a regulatory sequence of a promoter region of a gene and regulatory elements; it can also be used in the treatment and research of diseases, and many diseases are caused by single nucleotide mutation and deletion insertion mutation in the gene sequence, and the dual-function gene editing can provide a quick and useful solution for the treatment of such diseases. In a word, it is very necessary to develop a dual-functional gene editing system, which can have a broad application prospect in the aspects of disease treatment, animal model establishment and plant genetic breeding.
Brief Description of Drawings
Fig. 1: schematic representation of apodec 1-nCas9-UGI, apodec 3A-espbas91.1-UGI, apodec 3A-nCas9-UGI, and apodec 3A-Cas9-UGI constructs.
Fig. 2: comparing the frequency of C to T base substitutions of APOBEC 3A-eSPAS91.1-UGI, APOBEC3A-nCas9-UGI and APOBEC1-nCas9-UGI when using sgRNAs containing different length guide sequences. The data were derived from the OsCDC48 gene target site in rice protoplasts, and untreated protoplast samples were used as controls. Data from three independent biological replicates (n=3), each frequency (mean ± standard error) was calculated
Fig. 3: the efficiency of APOBEC3A-eSPCAs91.1-UGI to generate indels when using sgRNAs containing different length guide sequences was compared to that of pJIT-163-Ubi-Cas9 when using sgRNAs containing 20nt guide sequences. The data were derived from the OsCDC48 gene target site in rice protoplasts, and untreated protoplast samples were used as controls. Data from three independent biological replicates (n=3), each frequency (mean ± standard error) was calculated
Fig. 4: comparing the frequency of C to T base substitutions of APOBEC 3A-eSPAS91.1-UGI, APOBEC3A-nCas9-UGI and APOBEC1-nCas9-UGI when using sgRNAs containing different length guide sequences. The data were derived from the osnrt1.1b gene target site in rice protoplasts, and untreated protoplast samples were used as controls. Data from three independent biological replicates (n=3), each frequency (mean ± standard error) was calculated
Fig. 5: comparison of the efficiency of APOBEC 3A-eSPAS91.1-UGI and pJIT-163-Ubi-Cas9 in generating indels using sgRNAs containing different length guide sequences. The data were derived from the osnrt1.1b gene target site in rice protoplasts, and untreated protoplast samples were used as controls. Data from three independent biological replicates (n=3), each frequency (mean ± standard error) was calculated
Detailed Description
1. Definition of the definition
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. For example, standard recombinant DNA and molecular cloning techniques for use in the present invention are well known to those skilled in the art and are more fully described in the following documents: sambrook, j., fritsch, e.f., and Maniatis, t., molecular Cloning: ALaboratory Manual; cold Spring Harbor Laboratory Press: cold Spring Harbor,1989 (hereinafter "Sambrook"). Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
"genome" as used herein encompasses not only chromosomal DNA present in the nucleus of a cell, but also organelle DNA present in subcellular components of the cell (e.g., mitochondria, plastids).
As used herein, "organism" includes any organism suitable for genome editing, preferably eukaryotes. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cows, cats; poultry such as chickens, ducks, geese; plants include monocots and dicots such as rice, maize, wheat, sorghum, barley, soybean, peanut, arabidopsis, and the like.
By "genetically modified organism" or "genetically modified cell" is meant an organism or cell comprising within its genome an exogenous polynucleotide or modified gene or expression control sequence. For example, an exogenous polynucleotide can be stably integrated into the genome of an organism or cell and inherit successive generations. The exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. Modified genes or expression control sequences are those in which the sequence comprises single or multiple deoxynucleotide substitutions, deletions and additions in the genome of the organism or cell.
"exogenous" with respect to a sequence means a sequence from a foreign species, or if from the same species, a sequence that has undergone significant alteration in composition and/or locus from its native form by deliberate human intervention.
"Polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single-or double-stranded RNA or DNA polymers, optionally containing synthetic, unnatural or altered nucleotide bases. Nucleotides are referred to by their single letter designations as follows: "A" is adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.
"polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
As used herein, an "expression construct" refers to a vector, such as a recombinant vector, suitable for expression of a nucleotide sequence of interest in an organism. "expression" refers to the production of a functional product. For example, expression of a nucleotide sequence may refer to transcription of the nucleotide sequence (e.g., transcription into mRNA or functional RNA) and/or translation of RNA into a precursor or mature protein.
The "expression construct" of the invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, may be an RNA (e.g., mRNA) capable of translation.
The "expression construct" of the invention may comprise regulatory sequences of different origin and nucleotide sequences of interest, or regulatory sequences and nucleotide sequences of interest of the same origin but arranged in a manner different from that normally found in nature.
"regulatory sequence" and "regulatory element" are used interchangeably and refer to a nucleotide sequence that is located upstream (5 'non-coding sequence), intermediate or downstream (3' non-coding sequence) of a coding sequence and affects transcription, RNA processing or stability, or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
"promoter" refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment. In some embodiments of the invention, the promoter is a promoter capable of controlling transcription of a gene in a cell, whether or not it is derived from the cell. The promoter may be a constitutive or tissue specific or developmentally regulated or inducible promoter.
"constitutive promoter" refers to a promoter that will generally cause a gene to be expressed in most cases in most cell types. "tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to promoters that are expressed primarily, but not necessarily exclusively, in one tissue or organ, but also in one particular cell or cell type. "developmentally regulated promoter" refers to a promoter whose activity is determined by developmental events. An "inducible promoter" selectively expresses an operably linked DNA sequence in response to an endogenous or exogenous stimulus (environmental, hormonal, chemical signal, etc.).
Examples of promoters include, but are not limited to, polymerase (pol) I, pol II, or pol III promoters. Examples of pol I promoters include chicken RNA pol I promoters. Examples of pol II promoters include, but are not limited to, the cytomegalovirus immediate early (CMV) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, and the Simian Virus 40 (SV 40) immediate early promoter. Examples of pol III promoters include the U6 and H1 promoters. Inducible promoters such as the metallothionein promoter may be used. Other examples of promoters include the T7 phage promoter, the T3 phage promoter, the beta-galactosidase promoter, and the Sp6 phage promoter. When used in plants, the promoter may be the cauliflower mosaic virus 35S promoter, the maize Ubi-1 promoter, the wheat U6 promoter, the rice U3 promoter, the maize U3 promoter, the rice actin promoter.
As used herein, the term "operably linked" refers to a regulatory element (e.g., without limitation, a promoter sequence, a transcription termination sequence, etc.) linked to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcription regulatory element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art.
"introducing" a nucleic acid molecule (e.g., plasmid, linear nucleic acid fragment, RNA, etc.) or protein into an organism refers to transforming a cell of the organism with the nucleic acid or protein such that the nucleic acid or protein is capable of functioning in the cell. "transformation" as used herein includes both stable transformation and transient transformation.
"Stable transformation" refers to the introduction of an exogenous nucleotide sequence into the genome, resulting in stable inheritance of an exogenous gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generation thereof.
"transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell to perform a function without stable inheritance of an exogenous gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.
2. Dual function genome editing fusion proteins
The inventors have surprisingly found that the use of a genome editing fusion protein consisting of a CRISPR effector protein having nuclease activity and a deaminase enables simultaneous base editing and indels (indels) at different genomic loci of a cell by using guide RNAs of different lengths.
Thus, in a first aspect, the present invention provides a genome editing fusion protein comprising a CRISPR effector protein domain having nuclease activity and a deaminase domain.
As used herein, the term "CRISPR effector protein" generally refers to nucleases present in naturally occurring CRISPR systems, as well as modified forms thereof, variants thereof, catalytically active fragments thereof, and the like. The term encompasses any effector protein based on a CRISPR system that is capable of achieving gene targeting (e.g., gene editing, gene targeting regulation, etc.) within a cell.
By "CRISPR effector protein having nuclease activity" is meant that the CRISPR effector protein is capable of cleaving double stranded genomic DNA, thereby forming a Double Stranded Break (DSB).
Examples of "CRISPR effector proteins" include Cas9 nucleases or variants thereof. The Cas9 nuclease may be a Cas9 nuclease from a different species, such as spCas9 from streptococcus pyogenes(s) or SaCas9 derived from staphylococcus aureus (s.aureus). "Cas9 nuclease" and "Cas9" are used interchangeably herein to refer to an RNA-guided nuclease comprising a Cas9 protein or fragment thereof (e.g., a protein comprising the active DNA cleavage domain of Cas9 and/or the gRNA binding domain of Cas 9). Cas9 is a component of a CRISPR/Cas (clustered regularly interspaced short palindromic repeats and related systems) genome editing system that can target and cleave DNA target sequences to form DNA Double Strand Breaks (DSBs) under the direction of guide RNAs.
In some embodiments, the CRISPR effector protein having nuclease activity is derived from streptococcus pyogenes(s) Cas9. In some embodiments, the CRISPR effector protein having nuclease activity comprises the amino acid sequence set forth in SEQ ID No. 1 (SpCas 9). In some embodiments of the invention, the CRISPR effector protein having nuclease activity comprises the amino acid sequence set forth in SEQ ID NO. 2 (eSPCAS 9 (1.0)), SEQ ID NO. 3 (eSPCAS 9 (1.1)) or SEQ ID NO. 4 (SpCas 9-HF 1). In some preferred embodiments, the CRISPR effector protein having nuclease activity comprises the amino acid sequence set forth in SEQ ID NO. 3 (eSPCAs 9 (1.1)).
Examples of "CRISPR effector proteins having nuclease activity" may also include Cpf1 nucleases or variants thereof such as high specificity variants. The Cpf1 nucleases may be Cpf1 nucleases from different species, for example Cpf1 nucleases from Francisella novicida U, acidoaerococcus sp.BV3L6 and Lachnospiraceae bacterium ND 2006.
In some embodiments of the invention, the deaminase is a cytidine deaminase, e.g., an apolipoprotein B mRNA editing complex (apodec) family deaminase. The cytidine deaminase of the present invention is particularly a cytidine deaminase that can accept single-stranded DNA as a substrate. Examples of cytidine deaminase enzymes useful in the present invention include, but are not limited to: apodec 1 deaminase, activation-induced cytidine deaminase (AID), apodec 3G, APOBEC a or CDA1. In the present invention, cytidine deaminase in the fusion protein is capable of deaminating cytidine of single-stranded DNA generated in the formation of a fusion protein-guide RNA-DNA complex into U, and then effecting base substitution of C to T by base mismatch repair.
In some embodiments of the various aspects of the invention, the apodec 3A deaminase is a human apodec 3A deaminase. In some preferred embodiments, the human APOBEC3A deaminase comprises the amino acid sequence shown as SEQ ID NO. 5.
In some embodiments, the cytidine deaminase is located N-terminal to the CRISPR effector protein having nuclease activity.
In cells, uracil DNA glycosylase catalyzes the removal of U from DNA and initiates Base Excision Repair (BER), resulting in repair of U:G to C:G. Thus, without being limited by any theory, where the deaminase in the fusion protein is a cytidine deaminase, inclusion of uracil DNA glycosylase inhibitors in the genome editing fusion proteins of the present invention will be able to increase the efficiency of base editing.
Thus, in some embodiments of the invention involving the deaminase in the fusion protein being a cytidine deaminase, the genome editing fusion protein further comprises a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the uracil DNA glycosylase inhibitor comprises the amino acid sequence shown as SEQ ID NO. 6.
In some embodiments of the invention, the deaminase is adenine deaminase. Naturally occurring adenine deaminase converts adenosine on single stranded RNA to inosine (I) by deamination, with RNA as the substrate. Recently, by the directed evolution method, a DNA-dependent adenine deaminase capable of converting deoxyguanosine on a single-stranded DNA into inosine (I) with the single-stranded DNA as a substrate has been obtained based on tRNA adenine deaminase TadA of Escherichia coli. See Nicloe M.Gaudelli et al, doi: 10.1038/aperture 24644, 2017. In some embodiments, the deaminase is a DNA-dependent adenine deaminase.
In the present invention, the DNA-dependent adenine deaminase in the fusion protein is capable of deaminating adenosine of single-stranded DNA generated in the formation of the fusion protein-guide RNA-DNA complex into inosine (I), and substitution of A to G can be achieved by base mismatch repair since the DNA polymerase treats inosine (I) as guanine (G). Thus, where the deaminase in the fusion protein is a DNA-dependent adenine deaminase, one or more a bases in the genomic target sequence may be replaced with G bases.
In some embodiments of the invention, the adenine deaminase is a variant of the E.coli tRNA adenine deaminase TadA (ecTadA), particularly a variant that can accept single stranded DNA as a substrate. In some embodiments, the adenine deaminase comprises the amino acid sequence shown in SEQ ID NO. 7.
In some embodiments of the invention, the deaminase and the CRISPR effector protein having nuclease activity are fused by a linker. The linker may be a nonfunctional amino acid sequence 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or 20-25, 25-50) or more amino acids long, without secondary or higher structure. For example, the linker may be a flexible linker, such as GGGGS, GS, GAP, (GGGGS) x 3, GGS, and (GGS) x7, and the like. In some specific embodiments, the linker is an XTEN linker. In some embodiments, the linker is 32 amino acids long. In some specific embodiments, the amino acid sequence of the linker is: SGGSSGGSSGSETPGTSESATPESSGGSSGGS.
In some embodiments of the invention, the genome editing fusion protein of the invention further comprises a Nuclear Localization Sequence (NLS). In general, one or more NLS in the genome editing fusion protein should be of sufficient strength to drive the genome editing fusion protein in the nucleus of a cell to accumulate in an amount that can achieve its genome editing function. In general, the intensity of nuclear localization activity is determined by the number, location, one or more specific NLS(s) used, or a combination of these factors in the genome editing fusion protein.
In some embodiments of the invention, the NLS of the genome editing fusion protein of the invention may be located at the N-terminus and/or the C-terminus. In some embodiments, the genome editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLS. In some embodiments, the genome editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the N-terminus. In some embodiments, the genome editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the C-terminus. In some embodiments, the genome editing fusion protein comprises a combination of these, such as comprising one or more NLS at the N-terminus and one or more NLS at the C-terminus. When there is more than one NLS, each may be selected to be independent of the other NLS. In some preferred embodiments of the invention, the genome editing fusion protein comprises 2 NLS, e.g., the 2 NLS are located at the N-terminus and the C-terminus, respectively.
Generally, NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are also known. Non-limiting examples of NLS include: KKRKV, PKKKRKV, or SGGSPKKKRKV.
In addition, the genomic editing fusion proteins of the present invention may also include other targeting sequences, such as cytoplasmic targeting sequences, chloroplast targeting sequences, mitochondrial targeting sequences, etc., depending on the desired DNA location to be edited.
In some preferred embodiments, the genome editing fusion protein comprises the amino acid sequence shown in SEQ ID NO. 8.
3. Dual function genome editing system
In another aspect, the present invention provides a bifunctional genome editing system comprising a genome editing fusion protein of the present invention and/or an expression construct comprising a nucleotide sequence encoding a genome editing fusion protein of the present invention, and
i) At least one guide RNA for base substitution and/or an expression construct comprising a nucleotide sequence encoding said at least one guide RNA for base substitution, and/or
ii) at least one guide RNA for insertion and/or deletion and/or an expression construct comprising a nucleotide sequence encoding said at least one guide RNA for insertion and/or deletion.
As used herein, "gRNA" and "guide RNA" are used interchangeably to refer to an RNA molecule that is capable of forming a complex with a CRISPR effector protein and of targeting the complex to a target sequence due to some complementarity to the target sequence. In some embodiments of the invention, the guide RNA is a single stranded guide RNA (sgRNA). gRNA is typically composed of a scaffold sequence (scaffold) and a guide sequence (also known as a spacer sequence). The scaffold sequence of gRNA varies depending on its corresponding CRISPR effector protein. The skilled artisan is aware of the gRNA scaffold sequences required for different CRISPR effector proteins. The invention is not particularly limited to the scaffold sequence of the gRNA, which depends only on the CRISPR effector protein used. For example, for Cas9 (in particular spCas 9), the scaffold sequence of its sgrnas may be encoded by the following sequences: gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc tttt.
The guide sequence (also known as a spacer sequence) of a gRNA is a sequence identical or complementary to a target sequence, which achieves specific targeting by hybridization to the target sequence or its complement. For nuclease activity such as Cas9, the optimal gRNA guide sequence is 20 nucleotides in length.
The inventors have surprisingly found that when the gRNA leader is 20 nucleotides in length, the genome editing fusion proteins of the invention edit the target site primarily for insertions and/or deletions (indels); when the gRNA leader sequence is not 20 nucleotides in length, the genome editing fusion protein of the present invention is mainly base-substituted for editing of the target site. Thus, by introducing gRNAs having different leader sequence lengths, base substitutions and insertions and/or deletions (indels) can be introduced simultaneously and efficiently at the endogenous genomic locus widely.
In some embodiments, the guide RNA for insertion and/or deletion comprises a guide sequence of 20 nucleotides in length.
In some embodiments, the guide RNA for base substitution comprises a guide sequence of <20 or >20 nucleotides in length. In some preferred embodiments, the guide RNA for base substitution comprises a guide sequence of 19 nucleotides in length.
In some embodiments, the guide RNA for insertion and/or deletion and the guide RNA for base substitution target different genomic sites. For example, a guide RNA for insertion and/or deletion targeting a coding sequence of a gene for which a functional deletion is desired may result in an insertion and/or deletion in the coding sequence, thereby resulting in a functional deletion of the gene. Alternatively, the guide RNA for base substitution targets a target sequence that requires point mutation, which results in one or more base substitutions in the target sequence.
In order to obtain efficient expression in an organism, in some embodiments of the invention, the nucleotide sequence encoding the fusion protein is codon optimized for the organism to be edited.
Codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a more or most frequently used codon in the gene of the host cell (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons while maintaining the native amino acid sequence). Different species exhibit specific preferences for certain codons of a particular amino acid, codon preference (difference in codon usage between organisms) is often correlated with the translational efficiency of messenger RNAs (mRNA) which is believed to be dependent on the nature of the translated codons and availability of the particular transfer RNA (tRNA) molecules. The dominance of the selected trnas within the cell generally reflects the most frequently used codon for peptide synthesis. Thus, genes can be tailored to the optimal gene expression codon usage table in a given organism based on codon optimization can be readily obtained, e.g., at www.kazusa.orjp/codon/availability in the database ("35 a: available at 35, 16: nucleotide, 2000; and the same manner as those available at 2000; nucleotide, etc.).
In some embodiments of the invention, the nucleotide sequence encoding the genome editing fusion protein and/or the nucleotide sequence encoding the guide RNA is operably linked to an expression regulatory element, such as a promoter, in the expression construct.
In some embodiments, depending on the promoter used, the exact guide sequence of the sgrnas that can be used in the present invention is obtained by self-cleavage of tRNA (Zhang et al (2017) Genome Biology,2017, 18:191).
3. Method for producing genetically modified organisms
In another aspect, the invention provides a method of producing a genetically modified organism comprising introducing the bifunctional genome editing system of the invention into a cell of the organism.
In some embodiments, the guide RNA for insertion and/or deletion targets the genome editing fusion protein to at least one target sequence in the genome to be inserted and/or deleted. In some embodiments, the guide RNA for base substitution targets the genome editing fusion protein to at least one target sequence in the genome to be base substituted. In some embodiments, the guide RNA for insertion and/or deletion and the guide RNA for base substitution targets the genome editing fusion protein to at least one target sequence to be base substituted and at least one target sequence to be inserted and/or deleted in the genome.
In some embodiments, the method achieves insertion and/or deletion within at least one target sequence of the genome of the organism cell, while achieving base substitution within at least another target sequence of the genome of the organism cell.
In the present invention, the target sequence to be modified may be located at any position of the genome, for example, within a functional gene such as a protein-encoding gene, or may be located, for example, in a gene expression regulatory region such as a promoter region or an enhancer region, thereby effecting functional modification of the gene or modification of gene expression.
In some embodiments of the methods of the invention, screening for organisms, such as plants, having the desired nucleotide substitution is also included. Nucleotide substitutions in organisms such as plants can be detected by T7EI, PCR/RE or sequencing methods, see for example, shan, q., wang, y, li, J. & Gao, c.genome editing in rice and wheat using the CRISPR/Cas system.nat.protoc.9,2395-2410 (2014).
In the methods of the invention, the bifunctional genome editing system may be introduced into cells by various methods well known to those skilled in the art. Methods useful for introducing the genome editing system of the invention into cells include, but are not limited to: calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection, viral infection (e.g., baculovirus, vaccinia virus, adenovirus, adeno-associated virus, lentivirus, and other viruses), gene gun methods, PEG-mediated protoplast transformation, agrobacterium-mediated transformation.
Cells that can be genome edited by the methods of the invention can be from, for example, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks, geese; plants, including monocots and dicots, such as rice, maize, wheat, sorghum, barley, soybean, peanut, arabidopsis, and the like. In some preferred embodiments, the organism is a plant.
The method of the invention is particularly suitable for producing genetically modified plants, for example crop plants. In the methods of producing genetically modified plants of the invention, the bifunctional genome editing system can be introduced into plants by various methods well known to those skilled in the art. Methods that may be used to introduce the dual function editing system of the present invention into plants include, but are not limited to: gene gun method, PEG-mediated protoplast transformation, agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube channel method, and ovary injection method. Preferably, the bifunctional system is introduced into the plant by transient transformation.
In the method of the present invention, modification of the target sequence can be achieved by introducing or producing the bifunctional editing fusion protein and the guide RNA into a plant cell, and the modification can be stably inherited without stably transforming the bifunctional editing system into a plant. Thus avoiding the potential off-target effect of the stable bi-functional editing system and also avoiding the integration of the exogenous nucleotide sequence in the plant genome, thereby having higher biosafety.
In some preferred embodiments, the introducing is performed in the absence of selection pressure, thereby avoiding integration of the exogenous nucleotide sequence in the plant genome.
In some embodiments, the introducing comprises transforming the bifunctional genome editing system of the invention into an isolated plant cell or tissue, and then regenerating the transformed plant cell or tissue into a whole plant. Preferably, the regeneration is performed in the absence of selection pressure, i.e., without the use of any selection agent for the selection gene carried on the expression vector during tissue culture. The regeneration efficiency of plants can be improved without the use of a selection agent, resulting in modified plants that do not contain exogenous nucleotide sequences.
In other embodiments, the dual-function genome editing system of the present invention may be transformed into a specific location on an intact plant,
such as leaves, stem tips, pollen tubes, young ears or hypocotyls. This is particularly suitable for transformation of plants which are difficult to regenerate by tissue culture.
In some embodiments of the invention, the in vitro expressed protein and/or the in vitro transcribed RNA molecule is directly transformed into the plant. The protein and/or RNA molecules enable gene editing in plant cells, which are subsequently degraded by the cells, avoiding integration of the exogenous nucleotide sequence in the plant genome.
Thus, in some embodiments, genetic modification and breeding of plants using the methods of the invention can result in plants that are free of exogenous DNA integration, i.e., modified plants that are not transgenic (transgene-free). In addition, the dual function genome editing system of the present invention has high specificity (low off-target rate) when base editing is performed in plants, which also improves biosafety.
Plants that can be genetically edited by the methods of the invention include monocots and dicots. For example, the plant may be a crop plant, such as wheat, rice, maize, soybean, sunflower, sorghum, canola, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, tapioca, or potato.
In some embodiments of the invention, wherein the target sequence is associated with a plant trait, such as an agronomic trait, whereby the base editing results in the plant having an altered trait relative to a wild type plant. In the present invention, the target sequence to be modified may be located at any position of the genome, for example, within a functional gene such as a protein-encoding gene, or may be located, for example, in a gene expression regulatory region such as a promoter region or an enhancer region, thereby effecting functional modification of the gene or modification of gene expression.
In some embodiments of the invention, the method further comprises obtaining progeny of the genetically modified plant. In another aspect, the invention also provides a genetically modified plant or its progeny or part thereof, wherein the plant is obtained by the method of the invention as described above. In some embodiments, the genetically modified plant or its progeny or part thereof is non-transgenic.
In another aspect, the present invention also provides a plant breeding method comprising crossing a genetically modified first plant obtained by the method of the invention described above with a second plant that does not contain said genetic modification, thereby introducing said genetic modification into the second plant.
Examples
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1 efficiency of mutation of bifunctional Gene editing System in Rice protoplasts
Protoplasts used in the invention are derived from flowers 11 of japonica rice varieties.
To test the effect of editing endogenous genes by the dual-function editing system espcast 91.1-A3A, grnas of different lengths were designed at each of the target sites selected in rice genes OsCDC48 and osnrt1.1b (table 1). Using apodec 3A-nCas9-UGI, apodec 1-nCas9-UGI and wild-type SpCas9 as controls, following co-transformation of rice protoplasts with grnas, the efficiency of generating C to T base substitutions and indel mutations was analyzed using next generation sequencing technology (NGS).
TABLE 1 description of sgRNA target sites and sequences
| sgRNA | Targeting sequences |
| OsCDC48–sgRNA-17nt | CAGCCAGCGTCTGGCGCCGG |
| OsCDC48–sgRNA-18nt | CCAGCCAGCGTCTGGCGCCGG |
| OsCDC48–sgRNA-19nt | ACCAGCCAGCGTCTGGCGCCGG |
| OsCDC48–sgRNA-20nt | GACCAGCCAGCGTCTGGCGCCGG |
| OsCDC48–sgRNA-21nt | TGACCAGCCAGCGTCTGGCGCCGG |
| OsCDC48–sgRNA-22nt | CTGACCAGCCAGCGTCTGGCGCCGG |
| OsCDC48–sgRNA-23nt | GCTGACCAGCCAGCGTCTGGCGCCGG |
| OsCDC48–sgRNA-24nt | CGCTGACCAGCCAGCGTCTGGCGCCGG |
| OsNRT1.1B–sgRNA-15nt | TGGCGCCCGCGGCGGCGG |
| OsNRT1.1B–sgRNA-16nt | ATGGCGCCCGCGGCGGCGG |
| OsNRT1.1B–sgRNA-17nt | CATGGCGCCCGCGGCGGCGG |
| OsNRT1.1B–sgRNA-18nt | CCATGGCGCCCGCGGCGGCGG |
| OsNRT1.1B–sgRNA-19nt | GCCATGGCGCCCGCGGCGGCGG |
| OsNRT1.1B–sgRNA-20nt | GGCCATGGCGCCCGCGGCGGCGG |
Note that: PAM motifs in each target sequence are shown in bold.
The efficiency of C to T base editing and indel of the OsCDC48 gene target site in protoplasts was assessed using next generation sequencing technology (NGS). Finally, the APOBEC3A-eSPCAs91.1-UGI system was evaluated to have the highest C-to-T editing frequency at 19nt sgRNA length of 18.99% (FIG. 2). The positive control APOBEC3A-nCas9-UGI has the average editing efficiency of 35.03% at a sgRNA length of 19nt and the highest efficiency at a sgRNA length of 20nt, and the average editing efficiency of 42.56%.
Efficiency comparison to generate knockouts and/or insertions: the APOBEC3A-eSPCAs91.1-UGI system produced an indel editing frequency of 2.45% at a sgRNA length of 20nt (FIG. 3). Whereas wild-type pJIT-163-Ubi-Cas9 produced a maximum editing efficiency of 2.96% at a sgRNA length of 20nt (FIG. 3).
Likewise, next generation sequencing technology (NGS) was used to evaluate the efficiency of C to T base editing and indel of the osnrt1.1b gene target site in protoplasts. Finally, the highest editing frequency of the APOBEC 3A-eSPAS91.1-UGI system from C to T was 12.15% at a sgRNA length of 19nt (FIG. 4). The positive control APOBEC3A-nCas9-UGI has the average editing efficiency of 32.00% at a sgRNA length of 19nt and the highest efficiency at a sgRNA length of 20nt, and the average editing efficiency of 34.81%. The APOBEC3A-eSPCAs91.1-UGI system produced an indel editing frequency of 2.27% at a sgRNA length of 20nt (FIG. 5). Whereas wild-type pJIT-163-Ubi-Cas9 gave a maximum editing efficiency of 2.49% at a sgRNA length of 20nt (FIG. 5).
Considering comprehensively that APOBEC 3A-eSPAS91.1-UGI can be used as a dual-function system, when the sgRNA length is 19nt, high-efficiency C-to-T editing is generated, and the efficiency of generating indels is very low; the efficiency of indel generation is higher at a sgRNA length of 20nt, almost comparable to wild-type Cas9, and can be used to knock out genes. Multiple sgrnas of different lengths can be simultaneously transformed, and single base substitution and knockout and/or insertion of multiple genes can be simultaneously achieved as needed.
Claims (3)
1. A dual function genome editing system for genome editing of plant cells, the system comprising a genome editing fusion protein and/or an expression construct comprising a nucleotide sequence encoding the genome editing fusion protein, and
i) At least one guide RNA for base substitution and/or an expression construct comprising a nucleotide sequence encoding said at least one guide RNA for base substitution, and/or
ii) at least one guide RNA for insertion and/or deletion and/or an expression construct comprising a nucleotide sequence encoding said at least one guide RNA for insertion and/or deletion,
wherein the genome editing fusion protein comprises a CRISPR effector protein domain having nuclease activity and a deaminase domain,
wherein the guide RNA for base substitution is a 19 nucleotide long guide sequence;
wherein the guide RNA for insertion and/or deletion is a 20 nucleotide long guide sequence;
wherein the genome editing fusion protein comprises an amino acid sequence shown in SEQ ID NO. 8.
2. The method of claim 1, wherein the guide RNA for insertion and/or deletion and/or the guide RNA for base substitution targets the genome editing fusion protein to at least one target sequence to be base substituted and at least one target sequence to be inserted and/or deleted in the genome.
3. The method of claim 2, wherein said method achieves insertion and/or deletion within at least one target sequence of the genome of said plant cell and base substitution within at least another target sequence of the genome of said organism cell.
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