RU2794774C1 - Crispr/cas9 type ii genome editing system and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a type II CRISPR/Cas9 genome editing system, characterized in that the system contains the Cas9 protein, auxiliary proteins, crRNA (CRISPR RNA) and tracrRNA (transactivated CRISPR RNA) in the functional form of the RNP complex (ribonucleoprotein complex) Cas9 protein and guide RNA formed by hybridization of crRNA with tracrRNA.

EFFECT: invention is effective for DNA editing.

22 cl, 14 dwg, 11 ex

Description

The invention relates to a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium and relates to the field of genome editing technology.

BACKGROUND OF THE INVENTION

Gene editing technology allows you to modify the localization points of the DNA sequence. For example, first generation genome editing tools, zinc finger nucleases (ZFN), second generation genome editing tools such as effector nucleases like transcription activators (TALEN) can be used to transform target genomes. However, these methods are complex in design, difficult to manufacture, expensive and not universal.

The CRISPR (Regularly Clustered Short Palindromic Repeats)/Cas (CRISPR Associated Protein) system is an innate immune system derived from archaea and bacteria and serves as a third generation genome editing tool. Unlike previous genome editing tools (protein-DNA recognition), this method uses the principle of a complementary base pair of nucleic acids to determine the target DNA sequence and direct the Cas effector protein to perform site-specific cleavage, and is characterized by wide application, simple design, low cost and high efficiency. The Cas protein contains many different effector domains that are involved in various processes such as nucleic acid recognition, stabilization of complex structures, and hydrolysis of DNA phosphodiester bonds. Among them, the type II CRISPR/Cas9 system derived from Streptococcus pyogene Cas (SpCas9) has become the most widely used CRISPR/Cas system due to its high cleavage efficiency. This system identifies and cleaves the motif sequence adjacent to the protospacer (PAM), i.e. "NGG" on the target polynucleotide, leaving a blunt-ended overhang, affecting genome editing.

In large and diverse metagenomes containing uncultivated or even unknown microorganisms, there may be a large number of unexplored CRISPR/Cas9 systems, the activity of which in prokaryotes and eukaryotes, as well as in vitro media, requires confirmation.

In 2015, Dr. Byung-Chan Kim's team isolated a new anaerobic strain, AL017, from the faeces of C57BL/6J laboratory mice and analyzed the phylogenetic relationship of the strain to the 16SrRNA genetic sequence of prokaryotes and, in doing so, found that the strain is closely related to Holdemanellabiformis DSM 3989T, Faecalicoccus pleomorphus ATCC 29734T, Faecalitaleacylindroides ATCC 27803T and Allobaculumstercoricanis DSM 13633T (sequence homology is 87.4%, 87.3%, 86.9% and 86.9%, respectively). Based on numerous taxonomic data, this species is considered a new genus of the Erysipelothricaceae family and is named Faecalibaculum rodentium Gen.nov., sp.nov. Over the past five years, scientists around the world have been conducting research on this strain in two areas: gut microbial environment and high-fat diet, and gut microbial environment and tumorigenesis. However, no similar genome editing research has been reported.

BRIEF DESCRIPTION OF THE INVENTION

The aim of the invention is to overcome the shortcomings of the prior art and provide a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, where the genome editing system has novel physical and chemical properties and can identify many different PAM sequences (motif adjacent to protospacer), including NGTA and NNTA (N is A, C, G, or T).

To achieve the goal, the technical solution used in the invention is as follows.

Type II CRISPR/Cas9 genome editing system, characterized in that the system contains the Cas9 protein, accessory proteins, crPHK (CRISPR RNA) and tracrPHK (transactivated CRISPR RNA) in the functional form of the RNP complex (ribonucleoprotein complex) of the Cas9 protein and a guide RNA formed by hybridization of crRNA with tracrRNA;

wherein the Cas9 protein is a DNA endonuclease and the Cas9 protein has an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the amino acid sequence as shown in SEQ ID NO: 1.

The Cas9 protein cleaves double-stranded DNA complementary to crRNA upstream of the PAM sequence with a nuclease domain, where the nuclease domain is selected from an HNH-like nuclease domain, a RuvC-like nuclease domain, or a combination thereof.

Preferably, the type II CRISPR/Cas9 genome editing system is derived from Faecalibaculum rodentim.

In some embodiments, the term "ribonucleoprotein complex" preferably refers to "FrCas9 protein complex" in accordance with the invention.

Mutations at several key amino acid sites of the specific Cas9 protein are explained in detail as follows. Mutation of E to A at amino acid position 796 will result in the formation of the nickase nuclease. Mutation of N to A at amino acid position 902 will result in the formation of the nickase nuclease. Mutation of H to A at amino acid position 1010 will result in the formation of the nickase nuclease. A D to A mutation at amino acid position 1013 will result in the formation of the nickase nuclease.

Simultaneous mutation of E to A at amino acid position 796 and D to A at amino acid position 1013 will result in a Cas9 nuclease that is incapable of cleavage but retains binding capacity (ie dead Cas9).

Accessory proteins include Cas1 accessory protein, Cas2 accessory protein, and Csn2 accessory protein;

wherein the Cas1 accessory protein has an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% homology with the amino acid sequence as shown in SEQ ID NO: 2;

wherein the Cas2 accessory protein has an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% homology with the amino acid sequence as shown in SEQ ID NO: 3;

while the auxiliary protein Csn2 has an amino acid sequence,

as shown in SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% homology with the amino acid sequence as shown in SEQ ID NO: 4.

CRISPR RNA is generated by transcription of a CRISPR array and has an RNA sequence as shown in SEQ ID NO: 5, or an RNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the nucleic acid sequence. acids as shown in SEQ ID NO: 5; wherein the CRISPR array contains a direct repeat sequence and a spacer sequence, wherein the direct repeat sequence has a nucleic acid sequence as shown in SEQ ID NO: 6, or a nucleic acid sequence having at least 80%, 85%, 90%, 95 %, 98% or 99% homology to the nucleic acid sequence as shown in SEQ ID NO: 6; and wherein the spacer sequence has a nucleic acid sequence as shown in SEQ ID NO: 7, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% homology with the nucleic acid sequence as shown in SEQ ID NO: 7.

The transactivated CRISPR RNA contains a sequence complementary to the CRISPR RNA direct repeat sequence, and the transactivated CRISPR RNA has a nucleic acid sequence as shown in SEQ ID NO: 8, or a nucleic acid sequence having at least 70%, 80%, 85%, 90 %, 95%, 98% or 99% homology to the nucleic acid sequence as shown in SEQ ID NO: 8.

A guide RNA formed by hybridization of crRNA with tracrRNA has a scaffold consisting of a 7-24 nucleotide sequence of a crRNA direct repeat sequence and a tracrRNA sequence; preferably the two parts are fused with a "GAAA", "TGAA" or "AAAC" linker to form an sgRNA backbone; preferably an sgPHK framework formed by fusing a 20 nucleotide crRNA direct repeat sequence and a full-length tracrPHK sequence with "GAAA" is shown in SEQ ID NO: 9, and on this basis, preferably a highly efficient sgRNA framework variant is

the sgPHK framework formed by fusing the first 18 14 nucleotides of the crRNA direct repeat sequence and the last 69-65 nucleotides of tracrPHK with a linker sequence ("GAAA" etc.) is preferably selected from the following five frameworks:

(1) a 91 nucleotide long sgPHK framework that contains 18 nucleotides of the direct repeat sequence and 69 nucleotides of tracrPHK as shown in SEQ ID NO: 10;

(2) an 89 nt long sgPHK framework that contains 17 nt of direct repeat sequence and 68 nt of tracrPHK as shown in SEQ ID NO: 11;

(3) an 87 nt long sgPHK framework that contains 16 nt of direct repeat sequence and 67 nt of tracrPHK as shown in SEQ ID NO: 12;

(4) an 85 nt sgRNA backbone that contains 15 nt of direct repeat sequence and 66 nt of tracrPHK as shown in SEQ ID NO: 13;

(5) an 83 nucleotide long sgPHK framework that contains 14 nucleotides of the direct repeat sequence and 65 nucleotides of tracrPHK as shown in SEQ ID NO: 14;

optionally, the sgRNA framework has a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homology with any of SEQ ID NOs: 9-14.

The type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium binds or cleaves specific DNA in a biological process (e.g., genome editing process) by recognizing the complementary pairing of the guide RNA and target specific DNA, and the length of the paired binding portion of the guide RNA and the target specific DNA is 14 to 30 base pairs (preferably 20 to 23 base pairs); where specific DNA is prokaryotic or eukaryotic DNA.

The length of the paired binding portion of the guide RNA and target specific DNA is 21 base pairs, 22 base pairs, or 23 base pairs, with the RNP complex being highly sensitive to a base mismatch of 14 base pairs near the motif adjacent to the protospacer, and 14 base pairs is the initial region .

A protospacer-adjacent motif required for DNA binding or cleavage function is located 5'-NNTA-3' downstream of the guide RNA/sgRNA recognition sequence.

The invention also provides the use of the aforementioned type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium for DNA editing, CRISPR activation or interference. Preferably the DNA is prokaryotic or eukaryotic DNA.

As another aspect of the present invention, it further proposes the use of the above type II CRISPR/Cas9 genome editing system to produce a nickase, a dead Cas9, a base editor or a main editor. Preferably, the niqase, dead Cas9, base editor or base editor is produced in prokaryotes or eukaryotes.

Compared with the prior art, the invention provides the following beneficial effects.

(1) According to the invention, a CRSIPR/Cas9 type II genome editing system has been found in Faecalibaculum rodentium by bioinformatic analysis, said genome editing system has been applied to edit prokaryotic or eukaryotic genes, and provides a new opportunity to apply a genome editing toolkit.

(2) The invention provides a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, which has novel physical and chemical properties and can identify many different PAM sequences, wherein the specific PAM sequence recognized by the genome editing system is located at 5' -NNTA-3' below the RNA/sgPHK guideline recognition sequence.

(3) The invention provides a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, which has a high cleavage efficiency at the same DNA site compared to the most common SpCas9, while the target deviation is lower than that of the most common SpCas9, so it is a safer and more efficient genome editing tool.

(4) The invention provides a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, in which the PAM recognized at the level of the genome editing system has the characteristics of a palindrome, and therefore the targets are tail-to-tail distributed in the genome, which has more higher density than SpCas9.

(5) The invention provides a CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium, which is highly flexible and can be converted into a basic editor and a master editor, and is a genome editing tool that can be widely applied in various scenarios. .

BRIEF DESCRIPTION OF IMAGES

Figure 1 shows a compositional diagram of a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, in accordance with the invention.

Figure 2 is a structural diagram of the Cas9 protein of the type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, in accordance with the invention.

Figure 3 (3A-3F) shows an RNA secondary structure prediction scheme and an optimal sgRNA scaffold scheme for a guide RNA molecule recognized by the type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, in accordance with the invention. Figure 3A shows a full-length gRNA 95 nucleotides long. Figure 3B shows a 91 nucleotide long sgRNA backbone that includes 18 nucleotides of the direct repeat sequence and 69 nucleotides of tracrPHK. Figure 3C shows an 89 nt sgRNA backbone that includes 17 nt of the direct repeat sequence and 68 nt of tracrPHK. Figure 3D shows an 87 nt sgRNA backbone that includes 16 nt of the direct repeat sequence and 67 nt of tracrPHK. Figure 3E shows an 85 nt sgRNA backbone that includes 15 nt of the direct repeat sequence and 66 nt of tracrPHK. Figure 3F shows an 83 nt sgPHK backbone that includes 14 nt of the direct repeat sequence and 65 nt of tracrPHK.

Figure 4 is a diagram of the PAM prokaryotic sequences of the type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, in accordance with the invention.

Figure 5 is a schematic diagram of the prokaryotic interference experiments of the type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, in accordance with the invention.

Figure 6 is a schematic diagram of eukaryotic cleavage of a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, in accordance with the invention. Figure 6A is an ODN-PCR gel chart. Figure 6B is a Sanger sequence peak.

Figure 7 is a diagram showing the optimal length of sgRNA in the type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium according to the invention. Figure 7A is a graph showing the efficiency of sgRNA cleavage of various lengths at the HEK293 site of SITE2-T2. Figure 7B is a graph showing the efficiency of sgRNA cleavage of various lengths at the DNMT1-T3 site. Figure 7C is a graph showing the efficiency of sgRNA cleavage of various lengths at the RNF2-T6 site.

Figure 8 is a schematic diagram showing the optimal length of the sgRNA recognition sequence of a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium according to the invention.

Figure 9 is a schematic diagram showing a comparison of the targeting efficiency and off-targeting of GUIDE-seq in the type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium according to the invention compared to SpCas9.

Figure 10 is a diagram showing the experimental results of the basic editor FrCas9-BE; while Figure 10A is a schematic diagram and the results of confirming the shape of the FrCas9 nickase by PCR with a breakpoint ODN; Figure 10B is an edit box for nFrCas9-BE4Gram; Figure 10C is an edit window for nFrCas9-ABE7.10; Figure 10D is a Venn diagram of pathogenic mutations in the ClinVar database, which can be unambiguously adjusted by the base editors SpCas9 and FrCas9; Figure 10E shows the efficiency of C>T editing for FrCas9-BE4Gam with simultaneous use of 2 sgRNA tail-to-tail.

Figure 11 is a diagram showing the distribution of FrCas9 and SpCas9 targets in the human genome, with Figure 11A showing the distribution of 5'-GG-3' (representing SpCas9 PAM) in the human GRCh38 genome; Figure 11B shows the distribution of 5'-TA-3' (representing FrCas9 PAM) in the human GRCh38 genome.

Figure 12 shows the use of FrCa89-specific TATA box-targeted CRISPR interference and CRISPR activation, where Figure 12A is a schematic diagram of the TATA box of the ABCA1 gene targeted by FrCas9; Figure 12B shows CRISPR interference of FrCas9 and SpCas9 by targeting the TATA box and CRISPR activation of FrCas9 and SpCas9 by targeting the TATA box; Figure 12C shows CRISPR activation of FrCas9 and SpCas9 by targeting the TATA box.

Figure 13 shows a diagram of a genome editing system using Prime Editing technology.

Figure 14 shows the effect of editing the genome of the FrCas9-PE Primed Edit system verified by Sanger sequencing, where the measured sequence was CGAACACTCAAGGTAAT (SEQ ID NO: 33).

DETAILED DESCRIPTION OF THE INVENTION

In order to better explain the objects, technical solutions and advantages of the invention, the invention will be further described below with reference to specific embodiments.

The type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium includes the Cas9 protein, accessory proteins, CRISPR RNA and CRISPR transactivated RNA as shown in Figure 1. The Cas9 protein is a DNA endonuclease and has an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% homology with the amino acid sequence as shown in SEQ ID NO: 1.

As a preferred embodiment of the Faecalibaculum rodentium derived CRISPR/Cas9 type II genome editing system according to the invention, the Cas9 protein cleaves double-stranded DNA complementary to crRNA upstream of the PAM sequence with a nuclease domain as shown in Figure 2. The nuclease domain is selected from HNH-like nuclease domain or RuvC-like nuclease domain.

The Cas9 protein (Faecalibaculum rodentium Cas9), abbreviated as FrCas9 protein, contains 1372 amino acids (SEQ ID NO: 1) and is a multidomain and multifunctional DNA endonuclease. It effectively cleaves double-stranded DNA complementary to sgPHK upstream of the PAM sequence with a nuclease domain, for example, cleaves a DNA strand complementary to an sgPHK sequence with an HNH-like nuclease domain, or cleaves a non-complementary DNA strand with a RuvC-like nuclease domain. In this case, the mutation of E to A at position 796 of the amino acid will lead to the formation of the nickase nuclease; mutation of N to A at amino acid position 902 will result in the formation of nickase nuclease; mutation of H to A at amino acid position 1010 will result in the formation of nickase nuclease; a D to A mutation at amino acid position 1013 will result in the formation of the nickase nuclease; simultaneous mutation of E to A at amino acid position 796 and D to A at amino acid position 1013 will result in the formation of the Cas9 nuclease, which does not have the ability to cleave, but retains the ability to bind.

As a preferred embodiment of the Faecalibaculum rodentium derived CRISPR/Cas9 type II genome editing system according to the invention, the accessory proteins include the Cas1 accessory protein, the Cas2 accessory protein, and the Csn2 accessory protein. The Cas1 accessory protein has an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence as shown in SEQ ID NO: 2. Cas2 accessory protein has an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% homology with the amino acid sequence as shown in SEQ ID NO: 3. The Csn2 accessory protein has an amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99 % homology to the amino acid sequence as shown in SEQ ID NO: 4.

The accessory proteins Casl, Cas2 and Csn2 according to the invention are involved in exogenous gene uptake and crRNA maturation.

As a preferred embodiment of the Faecalibaculum rodentium derived CRISPR/Cas9 type II genome editing system according to the invention, CRISPR RNA is generated by transcription of the CRISPR array. CRISPR RNA has an RNA sequence as shown in SEQ ID NO: 5, or an RNA sequence having at least 80%, 85%, 90%, 95%, 98% or 99% homology with the nucleic acid sequence as shown in SEQ ID NO: 5. The CRISPR array contains a direct repeat sequence and a spacer sequence. The direct repeat sequence is shown in SEQ ID NO: 6. The spacer sequence is shown in SEQ ID NO: 7.

The CRISPR RNA (crPHK) according to the invention directs the Cas protein to recognize an invading foreign genome in base-complementary form. When bacteria are invaded by a bacteriophage or virus, a short segment of foreign DNA integrates as a new spacer between CRISPR spacer repeat sequences on the host chromosome, thereby providing a genetic record of the infection. When the foreign gene invades the organism again, the CRISPR array transcribes and produces a crRNA precursor (pre-crPHK) with a 30 bp spacer sequence at the 5' end, complementary to the sequence from the foreign gene. The 3' end is a 36 bp repeat sequence.

As a preferred embodiment of the Faecalibaculum rodentium derived CRISPR/Cas9 type II genome editing system according to the invention, the transactivated CRISPR RNA contains a sequence complementary to the CRISPR RNA direct repeat sequence as shown in SEQ ID NO: 8.

The transactivated crRNA (tracrPHK) according to the present invention is an RNA that is not encoded by a protein and is involved in the maturation of crRNA and the formation of sgRNA. Under the action of tracrPHK and Cas9 nuclease, pre-crPHK removes from 0 to 16 nucleotides upstream of the spacer sequence and from 12 to 29 nucleotides downstream of the repeat sequence to form mature crRNA and binds tracrPHK to form the tracrPHK-crPHK complex, which contains a portion that recognizes a foreign DNA sequence, and has a length of 14 to 30 base pairs. A tetraloop (e.g., the sequence "GAAA", "TGAA", or "AAAC") of four bases can be added between the lower end of crPHK and the upper end of tracrPHK to bind tracrPHK and crPHK to form an sgPHK containing two convex and three duplex structures in direction towards the upper end and a hairpin structure towards the lower end, which can be further divided into a part that recognizes a foreign DNA sequence and a scaffold part.

Endonuclease digestion can be further optimized by adjusting the length of the foreign DNA sequence recognition portion of sgPHK and the length of tracrPHK. Early experiments of the present invention prove that the optimal length of the sgRNA portion that recognizes an exogenous DNA sequence is 21 base pairs, 22 base pairs, or 23 base pairs (as shown in Figure 8), and the optimal length of the framework portion is one of the following 5 types, as shown in Figures 3 and 7: 91 nt (18 nt crPHK direct repeat+69 tracrPHK nt), 89 nt (17 nt crPHK direct repeat+68 tracrPHK nt), 87 nt (16 crPHK direct repeat+67 nt). tracrPHK), 85 nt (15 nt crRNA direct repeat + 66 tracrPHK nucleotides), and 83 nt (14 crRNA direct repeat nt + 65 tracrPHK nucleotides).

As a preferred embodiment of the Faecalibaculum rodentium derived CRISPR/Cas9 type II genome editing system according to the invention, the Faecalibaculum rodentium derived CRISPR/Cas9 type II genome editing system binds or cleaves DNA structures involved in the genome editing process.

As a preferred embodiment of the type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, according to the invention, the DNA is prokaryotic or eukaryotic DNA.

In some embodiments, FrCas9 of the present invention can recognize a plurality of DNA double-strand breaks (DSBs) adjacent to a protospacer (PAM) immediately below the targeting sequence. The FrCas9 protein requires two important factors to recognize a targeting sequence: one is a nucleotide complementary to the crRNA spacer; and the other is a protospacer adjacent motif (PAM) sequence adjacent to a complementary sequence. The depletion experiment shows that FrCas9 has a cleavage effect in the prokaryotic system and tentatively verifies the third and fourth positions of the PAM sequence recognized by this newly discovered type II CRISPR/Cas9 system, which are TA, as shown in Figure 4. Further confirmed by experiment on interference that the PAM downstream of the targeting sequence recognized by FrCas9 is 5'-NNTA-3' as shown in Figure 5. All of the above PAMs were verified by eukaryotic experiment as shown in Figure 6. By artificially constructing a spacer sequence in crRNA, this CRISPR-Cas9 system can target almost all DNA sequences in the genome of interest, producing a site-specific blunt-ended double-strand break (DSB). DSB repair with non-homologous ends results in small random insertions/deletions (indels) at the cleavage site to inactivate the gene of interest. Alternatively, with high fidelity homologous repair, precise genomic modifications at the DSB site can be performed using homologous repair templates.

Most human genetic diseases are single base mutations that are not treatable by conventional means. The base editor is one of the newest and most efficient ways to precisely edit the genome. It is characterized by the use of a break-shaped CRISPR-Cas protein to locate specific target DNAs and the use of DNA deaminase to modify and mutate this target DNA to repair damaged bases without generating double-stranded DNA fragmentation. In some embodiments, the combination of E796A nFrCas9 with the optimized fourth generation cytidine base editor BE4Gam can successfully construct the E796A nFrCas9-BEGam cytosine base editor (CBE) such that the C:G base pair in DNA can be mutated to T:A. The combination of E796A nFrCas9 with the 7th generation adenine base editor ABE7.10 can successfully construct the E796A nFrCas9-BABE7.10 adenine base editor (ABE) so that the DNA A:T base pair can be mutated to G:C.

In some embodiments, the FrCas9 specific PAM of the present invention is an NNTA specifically targeted to the TATA box (one of the elements that make up the eukaryotic promoter), such that FrCas9 specifically targets the TATA box to create a unique CRISPR interference effect (CRISPRi)/ CRISPR activation (CRISPRa). Of these, CRISPRi can be achieved by directly targeting the cleavage of the TATA box with the active form of FrCas9 and destroying the TATA box, or by direct binding to the TATA box with dFrCas9 (i.e., dead Cas9) without performing cleavage. CRISPRa can be achieved, among other things, by targeting dFrCas9-VP64 to the site of the TATA box.

Prime Editing (PE) is a brand new tool for precise genome editing. With this technology, free single base substitution and precise insertion and deletion of multiple bases can be realized, greatly reducing the harmful by-products created by indels in the genome editing process and greatly improving editing accuracy. It is generally accepted that this is a significant achievement in genome editing. In some embodiments, the FrCas9 of the present invention is fused to a reverse transcriptase and the corresponding regRNA can be engineered according to its PAM sequence to create an FrCas9-PE genome editing system. In particular, two sequences are added to the regPHK sequence at the 3' end. The first sequence is a primer binding site (PBS) that can be complementary to the end of a broken target DNA strand to initiate the reverse transcription process, and the second sequence is a reverse transcription template (RT template) that performs a target point or insertion mutation. deletions to achieve precise genome editing.

Yet another object of the invention is to provide the use of a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium for prokaryotic or eukaryotic gene editing, CRISPR activation or CRISPR interference.

As a preferred application according to the invention, a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium is used to bind or cleave DNA structures involved at the DNA level.

Example 1

In the example, the RNA secondary structure of the RNA guide molecule recognized by the CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium according to the invention was predicted, the RNA structure after tracrPHK transcription and repeat was predicted by modeling the two RNA binding process, the resulting secondary the RNA structure is shown in Figure 3.

(1) Materials: predicted tracrRNA and repeat sequences, and predicted anti-repeat sequences.

(2) Software: NUPACK (http://www.nupack.org/partition/new)

(3) Prediction method: The interaction process of 1 µl of each of two RNAs at 37°C in vitro was simulated using the NUPACK online application, as a result, the secondary structure of the resulting RNA composition was predicted to obtain the secondary structure of RNA, as shown in Figure 2.

As shown in Figure 3, pre-crRNA removed 0 to 16 nucleotides upstream of the spacer sequence and 12 to 29 nucleotides downstream of the repeat sequence by tracrPHK and Cas9 nuclease to form mature crRNA, which fused to tracrPHK to form the tracrPHK-crPHK complex. containing an exogenous DNA sequence complementary to the spacer sequence, while the length of the sequence ranged from 14 to 30 base pairs. They were linked to form sgRNA by adding a four-base tetraloop between the lower end of the crRNA and the upper end of the tracrRNA, containing two convex and three duplex structures towards the upper end and three hairpin structures towards the lower end.

Example 2

In an example, the protospacer adjacent motif (PAM) recognized by the CRTSPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium is 5'-NNTA-3' in the prokaryotic system.

(1) Materials: genes related to the CRISPR/Cas9 genome editing system predicted by the implementation described above.

(2) Inspection method: In this example, a prokaryotic inspection system was constructed with respect to the CRISPR/Cas9 type II genome editing system obtained from Faecalibaculum rodentium according to the invention to test the effect of its cleavage and preliminary study of the recognized PAM sequence by the second generation of technology sequencing, with the result shown in Figure 4.

Detailed protocols are given below.

(a) The CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium (comprising Cas9 endonuclease, Casl, Cas2, Csn2 accessory proteins, CRISPR array and tracrPHK non-coding RNA) according to the invention was inserted into the pACYC184 vector, where the Cas9 protein was subjected to Escherichia coli codon optimization. The natural spacer sequences and the spacer sequences in the library were added to the CRISPR array, and the strong heterologous J23119 promoter was added to the Cas9 protein and the CRISPR array to construct the pACYC184-FrCas9 prokaryotic expression plasmid.

(b) Seven random bases (total 16,384 inserts) were added to the 3' spacer sequence of the library. The two restriction endonuclease sites EcoRI and Ncol were selected from the polyclonal MCS vector pUC19. The library was cloned into a vector and a target library plasmid was constructed.

(c) A plasmid containing pACYC184-FrCas9 or empty pACYC184 and a target library was coelectrically transfected into E. coli DH5a. After restoration of functions at 25°C for 2 h, they were evenly distributed in SOB medium, which has dual resistance to ampicillin sodium (100 μg/ml) and chloramphenicol (34 μg/ml) for incubation at 25°C for 30 hours, at The plasmid was then harvested by alkaline lysis.

(d) PCR amplification was performed on a site containing a spacer sequence and seven random bases. Secondary sequencing was performed at the two ends of the PCR product by adding a linker. PAM depletion value (PPDV) was calculated relative to the no-load control group. The PAM sequence of the CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium was generated using Weblogo, where the PAM sequence is 5'-NNTA-3'.

Figure 3 is a schematic diagram of a conserved PAM sequence recognized by the Faecalibaculum rodentium derived CRISPR/Cas9 type II genome editing system according to the invention in a prokaryotic system. To calculate the PAM depletion value (PPDV) relative to the no-load control group, a second-generation sequencing analysis of the DNA library obtained from the depletion experiment was performed. The FrCas9 PAM sequence generated by Weblogo is 5'-NNTA-3'.

Example 3

In the example, the motif adjacent to the protospacer (PAM) recognized in the CRISPR/Cas9 type II genome editing system obtained from Faecalibaculum rodentium according to the invention was verified by means of an interference experiment, and its cleavage ability at the prokaryotic level and potential ability to edit the genome in eukaryotes. The results of the interference experiments are shown in Figure 5, with schematic diagrams of various possible PAM sequences recognized by the type II CRISPR7Cas9 genome editing system derived from Faecalibaculum rodentium according to the invention shown in Figure 5.

(1) Materials: pACYC184-FrCas9, target library plasmid prepared in Example 2, and pre-recognized PAM.

(2) Test method: In this example, the protospacer adjacent motif (PAM) recognized by the CRISPR/Cas9 type II genome editing system obtained from Faecalibaculum rodentium according to the invention in the prokaryotic system was further determined by an interference experiment.

Detailed protocols are given below.

(a) A total of 16 combined sequences were generated by adding NNTA at position 3' of the spacer sequence, and the target plasmid was constructed by cloning into pUC19 through the EcoRI and NcoI endonuclease restriction sites, respectively.

(b) 16 target plasmids, respectively, were transfected into E. coli DH5a electrogenic competence containing FrCas9-related loci, with the plasmids gradually diluted after recovery at 25° C. for 2 hours. Target id plasmas were incubated overnight at 25° C. in SOB media dually resistant to ampicillin sodium (100 μg/mL) and chloramphenicol (34 μg/mL) by dot blotting to determine the number of monoclonal bacteria.

Figure 5 is a schematic diagram of an interference experiment of a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium in accordance with the invention. Based on the NNTA found in the depletion experiment, 16 target plasmids were constructed with different combinations of NN. The number of monoclonal colonies was observed by means of an interference experiment. In Figure 5A, the leftmost column was labeled single FrCas9 and the right side was labeled the target plasmid targeted by FrCas9 cleavage. The results showed that the number of colonies in the right column decreased. Figure 5B is a statistical diagram of the cleavage effects of multiple PAM sequences recognized by the CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium according to the invention and illustrates that the number of colony forming units (KOE/CFU) of target plasmids of 14 different combinations of NN reduced compared to the control group. The above results showed that the protospacer adjacent motif (PAM) recognized by the newly discovered CRISPR/Cas9 system in the prokaryotic system by interference experiment was 5'-NNTA-3' (N is any base selected from the group consisting of from A, T, C and G).

Example 4

In the example, the range of tracrRNA required for cleavage of target DNA sequences in the Faecalibaculum rodentium derived CRISPR/Cas9 type II genome editing system according to the invention was verified by interference experiments, and the results of the interference experiments are shown in Figure 5C.

(1) Materials: pACYC 184-FrCas9, target plasmid, previously recognized by PAM prepared in Example 2.

(2) Verification Method: In this example, the range of tracrRNA required for cleavage of the target DNA sequence was further determined using the Faecalibaculum rodentium-derived CRISPR/Cas9 type II genome editing system in a prokaryotic system.

Detailed protocols are given below.

(a) A total of six gene sequences of all possible non-coding regions in wild-type Faecalibaculum rodentium were cloned into the target plasmid by Gibson cloning to construct target plasmids NC 1 to 6, where NC6 is generated by splicing the full length of the non-coding regions, and NC 1 to 5, respectively, represent the possible non-coding regions of segments one through five. A strong heterologous J23119 promoter was added to each NC plasmid towards the top of the non-coding region, where "+" was the forward non-coding region and "-" was the reverse non-coding region, respectively;

(b) From pACYC184-FrCas9 obtained in Example 2, all possible non-coding regions were removed by the PCR homologous recombination method, and the CRISPR-related protein and CRISPR array gene sequence were preserved, thus constructing pACYC184-ΔFrCas9;

(c) 12 target plasmids, respectively, were transfected into E. coli DH5a electrogenic competence containing pACYC184-ΔFrCas9, with the plasmids gradually diluted after recovery at 25° C. for 2 hours. Target plasmids were incubated overnight at 25° C. in SOB media dually resistant to ampicillin sodium (100 μg/mL) and chloramphenicol (34 μg/mL) by dot blotting to determine the number of monoclonal bacteria.

Figure 5C is a schematic diagram of an interference experiment of the type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium according to the invention, the results show that the monoclonal colonies of NC1 and NC6 were significantly smaller than the colonies of NC from 2 to 5, indicating that an alternative non-coding region in the first segment may assist FrCas9 in efficient and targeted cleavage of DNA sequences in E. coli.

Example 5

In an example, the ability to cleave target DNA sequences in eukaryotic cells in a type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium was verified through an ODN experiment. The ODN-PCR result is plotted in Figure 6A and the Sanger sequencing result is plotted in Figure 6B.

(1) Materials: All amino acid sequences, CRISPR template sequences, tracrPHK sequences, and the recognized PAM of the Faecalibaculum rodentium edited gene obtained in Examples 1-4.

(2) Verification Method: In this example, the ability to cleave target DNA sequences in eukaryotic cells was verified by ODN experiment in the CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium.

Detailed protocols are given below.

(a) An optimized human FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to create the PX330-FrCas9 plasmid.

(b) 30 base pairs upstream of the NGTA, NATA, NCTA and NTTA of the human RNF2 gene were chosen as the target sequence. The CRISPR array was constructed using the Gibson method. The strong human eukaryotic U6 promoter was added upstream of the CRISPR template to construct the PX330-FrCas9 template plasmid.

(c) A mouse-derived eukaryotic strong U6 promoter was added upstream of the tracrRNA shown in Example 4 to construct the PX330-FrCas9-array-tracrRNA plasmid.

(d) PX330-FrCas9-array-tracrRNA targeting various gene sites, constructed as above with 2.5 μg and 1.5 μl ODN, was electrically transfected into HEK293T cells in good condition. All cells were harvested after 72 hours for DNA extraction.

(e) ODN-PCR was performed by designing a primer pair adjacent to the RNF2 target gene site and on the ODN sequence, and agarose gel electrophoresis was performed to observe the presence of a band to preliminarily determine whether target cleavage occurred or not.

(f) Successful insertion of ODN into the target site was verified by Sanger sequencing and FrCas9 was confirmed to have the ability to edit target DNA in eukaryotic cells.

The results of ODN-PCR are shown in Figure 6A. NGTA, NATA, NCTA and NTTA had target strips of the correct size. As shown in Figure 8 with Sanger sequencing results, the position of the ODN insert occurred 3-4 base pairs towards the upper end of the PAM, indicating that the cleavage range of the target gene sequence in FrCas9 was consistent with that of previous SpCas9.

Example 6

In the example, the optimal length of the sgRNA recognition portion in eukaryotic cells in the CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium according to the invention was verified by ODN experiment, and the results are shown in Figure 7.

(1) Materials: all amino acid sequences, sgPHK sequences and recognized PAMs of the edited Faecalibaculum rodentium gene obtained in Examples 1-4;

(2) Verification Method: In this example, the optimal length of the sgRNA recognition portion in eukaryotic cells in the CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium was verified by ODN experiment.

Detailed protocols are given below.

(a) An optimized human-derived FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.

(b) A target sequence 30 base pairs upstream of GGTA near the SpCas9 target site was chosen as the targeting sequence, which was found in a previous study to have a good cleavage effect, with sgRNA with a recognition length of 19 to 23 pairs bases was constructed according to the Gibson method. The strong human eukaryotic U6 promoter was added towards the upper end of the sgRNA to construct the PX330-FrCas9-sgRNA plasmid.

(c) PX330-FrCas9-sgRNA targeting different gene sites, constructed as above using 2.5 μg and 1.5 μl ODN, was electrically transfected into HEK293T cells in good condition. All cells were harvested after 72 hours for DNA extraction.

(d) ODN-PCR was performed by designing a pair of primers adjacent to the target gene site and on the ODN sequence, and agarose gel electrophoresis was performed to observe the presence of a band to preliminarily determine whether target cleavage occurred or not and compare intensity of sgRNA bands with recognition length from 19 to 23 base pairs.

(e) A high-throughput Amplicon library was generated to quantify inserts in the target region, comparing the effects of sgRNA cleavage with a recognition length of 19 to 23 base pairs.

As shown in Figure 7, the optimal sgRNA recognition length for FrCas9 is 21 bp, 22 bp, or 23 bp.

Example 7

In the example, the optimal sgRNA backbone length in eukaryotic cells in the Faecalibaculum rodentium derived CRISPR/Cas9 type II genome editing system according to the invention was verified by ODN experiment and the results are shown in Figure 8.

(1) Materials: all amino acid sequences, sgPHK sequences and recognized PAMs of the edited Faecalibaculum rodentium gene obtained in Examples 1-6.

(2) Verification Method: In this example, the ODN experiment was used to verify the optimal length of the eukaryotic cell sgRNA framework in the type II CRISPR7Cas9 genome editing system derived from Faecalibaculum rodentium.

Detailed protocols are given below.

(a) An optimized human-derived FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.

(b) A target sequence 30 base pairs upstream of GGTA near the SpCas9 target site was chosen as the targeting sequence, which was found in a previous study to have a good cleavage effect, with sgRNA with a framework length of 71 nucleotides to 95 nucleotides was constructed according to the Gibson method. The strong human eukaryotic U6 promoter was added upstream of sgRNA to construct the PX330-FrCas9-sgRNA plasmid.

(c) The plasmid PX330-FrCas9-sgRNA, targeting various gene sites, constructed as above using 2.5 μg and 1.5 μl of ODN, was electrically transfected into HEK293T cells in good condition. All cells were harvested after 72 hours for DNA extraction.

(d) ODN-PCR was performed by designing a primer pair near the target gene site and on the ODN sequence, and agarose gel electrophoresis was performed to observe the presence of bands to preliminarily determine whether the target was cleaved or not and compare the intensity sgRNA bands with a framework length from 71 nucleotides to 95 nucleotides.

(e) A high-throughput Amplicon library was generated to quantify inserts in the target region, comparing the effects of sgRNA cleavage with a framework length of 71 nt to 95 nt.

As shown in Figures 8 and 3, the optimal sgRNA backbone for FrCas9 is 83 to 91 nucleotides.

Example 8

In the example, the cleavage effect in eukaryotic cells in the CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium was verified through an ODN experiment to be higher than that of SpCas9, and the off-target rate was lower than that of SpCas9.

(1) Materials: all amino acid sequences, 22 bp sgPHK sequence, 5'-GGTA-3' PAM sequence, SpCas9 amino acid sequence, SpCas9 sgPHK sequence and SpCas9 5'-NGG-3' PAM sequence of edited Faecalibaculum rodentium gene obtained in examples 1-6;

(2) Verification method: In this example, the GUIDE-seq experiment was used to verify that the cleavage effect in the nucleated cells of eukaryotic cells of the CRISPR/Cas9 type II genome editing system derived from Faecalibaculum rodentium according to the invention was higher than that of SpCas9 and the off-target frequency was lower than that of SpCas9.

Detailed protocols are given below.

(a) An optimized human-derived FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.

(b) A target sequence 30 base pairs upstream of GGTA near the target site of SpCas9 was chosen as the targeting sequence, which was found to have a good cleavage effect in a previous study, and an sgRNA with a recognition length of 22 base pairs was constructed by the Gibson method. The strong human eukaryotic promoter U6 was added upstream of sgPHK to construct the PX330-FrCas9-sgPHK plasmid and, concurrently, construct the PX330-SpCas9-sgPHK plasmid from the 20 bp sgRNA SpCas9.

(c) Plasmid PX330-FrCas9-sgRNA and plasmid PX330-SpCas9-sgRNA targeting different gene sites, constructed as above with 2.5 μg and 1.5 μl of ODN, were electrically transfected into HEK293T cells in good condition . All cells were harvested after 72 hours for DNA extraction.

(d) ODN-PCR was performed by designing a primer pair adjacent to the target gene site and on the ODN sequence, while agarose gel electrophoresis was performed to observe the presence of bands to preliminarily determine whether the target was cleaved or not, and from the DNA bands created the GUIDE-seq library.

(e) Using bioinformatics analysis, the effects of target cleavage and detargeting of SpCas9 and FrCas9 at the same site were compared.

As shown in Figure 9, FrCas9 was at a target read value of 3257 at the DYRK1A-T2 site, which is higher than SpCas9 at a target read value of 2456. Meanwhile, no off-target for FrCas9 was detected, while SpCas9 showed off-target at three sites. FrCas9 was at a target read value of 34970 at the GRIB2B-T9 site, which was higher than SpCas9 at a target read value of 20434. three sites. The above data showed that FrCas9 was a Cas9 protein superior to SpCas9 in cleavage efficiency and specificity.

Example 9. FrCas9 can be used for Prime Editing (PE)

Most human genetic diseases are associated with mutations that are not amenable to conventional treatment. The base editor is one of the newest and most efficient ways to precisely edit the genome. It is characterized by the use of a break-shaped CRISPR-Cas protein to locate specific target DNAs and the use of DNA deaminase to modify and mutate this target DNA to repair damaged bases without generating double-stranded DNA fragmentation. The cytosine base editor (CBE) mutated the C:G DNA base pair to T:A, and the adenine base editor (ABE) mutated the A:T base pair to G:C.

First, three point mutations, E796A, H1010A, and D1013A, respectively, were incorporated to produce different FrCas9 (nFrCas9) nicases. E796A nFrCas9 were then combined with the optimized fourth generation cytidine base editor BE4Gam and the seventh generation adenine base editor ABE7.10. It was observed that the FrCas9-BE4Gam edit window was 6-10th bases (Figure 10B) and the FrCas9-ABE7.10 window was 6-8th bases (Figure 10C). Based on the above characteristics, the targeting regions of FrCas9-BE4Gam and FrCas9-ABE7.10 were calculated in the ClinVar databases. For pathogenic mutations that could be accurately corrected for by FrCas9-BE4Gam, 90.38% (235/260) of the unique events were different from SpCas9-BE4Gam. For pathogenic mutations that could be fine-tuned by FrCas9-ABE7.10, 92.21% (1196/1297) of unique events were different from SpCas9-ABE7.10 (Figure 10D). Thus, TA-enriched PAM FrCas9 significantly expanded the targets in the human genome for the base editor to correct human disease-associated mutations.

In addition, PAM FrCas9 (5'-NNTA-3') was palindromic, suggesting tail-to-tail pairing of sgRNA (Figure 10E). This feature can enhance the capabilities of the basic FrCas9 editors by simultaneously changing two close editing windows (Figure 10E) and increase the distribution of targets and the density of FrCas9 sgPHK. The distribution of 5'-GG-3' (represented for SpCas9 PAM) and 5'-TA-3' (represented for FrCas9 PAM) in human genomes was calculated (Figures 11A and 11B). Compared to SpCas9 (median=5 bp, mean=8.66 bp), FrCas9 showed a more intense distribution (median=1 bp, mean=6.16 bp) in human genomes, providing additional applicable loci.

Example 10 FrCas9 can target the TATA box to modulate gene expression as an effective tool for activating and inhibiting CRISPR.

TATA-box (TATA-box/Hogness box) is one of the elements that make up the eukaryotic promoter. Its sequential order is TATA(A/T)A(A/T) (non-matrix sequence of strings). It is located about -30 base pairs (-25 to -32 base pairs) upstream of the transcription initiation point of most eukaryotic genes and is primarily composed of A-T base pairs, which is a selection criterion for determining the transcriptional initiation of genes. Transcription can begin only after strong binding to the TATA box of one of the RNA polymerase binding sites. Because the FrCas9 PAM sequence is NNTA, it has natural advantages when targeting the TATA box.

FrCas9 CRISPR interference (CRISPRi) was tested in three TATA box promoted genes, ABCA1, UCP3 and RANKL (Figure 12A). By cleaving the TATA box, FrCas9 reduced the expression of ABCA1, UCP3, and RANKL by 31.37%, 49.91%, and 39.62%, respectively. At the same time, dFrCas9 was also used for direct binding to the TATA box without cleavage, while the expression of ABCA1, UCP3 and RANKL decreased by 61.67%, 45.61% and 42.60% due to direct binding of dFrCas9 to TATA -box, respectively (Figure 12B). Thus, FrCas9 has a unique potential for efficient genome engineering of genetic diseases associated with the TATA box.

In addition, FrCas9 CRISPR activation (CRISPRa) was tested using dFrCas9-VP64 directly targeted to the TATA box and compared with dSpCas9-VP64 targeted upstream from the TATA box. CRISPRa experiments were carried out with the ABCA1, SOD1, GH1, and BLM2 genes. The results showed that dFrCas9-VP64 provided efficient transcriptional activation. Moreover, the activation fold of dFrCas9-VP64 in ABCA1, GH1 and BLM2 was higher than that of dSpCas9-VP64, and the activation fold of the SOD1 gene was comparable to that of dSpCas9-VP64 (Figure 12C). Thus, FrCas9 is a promising tool for CRISPR screening due to its unique 5'-NNTA-3' PAM.

Example 11. FrCas9 can be used for Prime Editing (PE)

Primed Editing (PE) is a completely new tool for precise gene editing. This technology has the potential to significantly reduce the amount of harmful by-products generated by indels in the gene editing process, greatly improve the accuracy of editing, and potentially overcome fundamental barriers to treating genetic diseases with existing gene editing techniques. The PE system consists of two components such as an engineered guide RNA (regPHK) and a primary editor protein. regPHKs perform a dual function: they have the ability to direct the edited protein to the target site and contain the edited template sequence. The editor-in-chief protein consists of a mutated Cas9 nickase (which breaks only one strand of DNA) and a fused reverse transcriptase. After Cas9 cleaves the target site, reverse transcriptase uses regPHK as a template for reverse transcription, thereby making the required editing in the DNA strand, with the corrected sequence preferentially replacing the original genomic DNA, thereby permanently editing the target site (Figure 13 ).

Based on the biological characteristics of FrCas9, FrCas9 was fused with reverse transcriptase, the corresponding regPHK was constructed according to its PAM sequence, the FrCas9-PE gene editing system was created, and its gene editing efficiency was optimized. regRNA was constructed according to the PAM sequence of FrCas9. Compared to sgPHK, the regPHK sequence has two additional sequences at the 3' end. The first sequence is a primer binding site (PBS), which may be complementary to the end of the broken strand of the target DNA to initiate reverse transcription. The second sequence is a reverse transcription template (RT template) that performs target point mutations or indel mutations to achieve precise gene editing. Previous studies have shown that the length of the PBS and RT template sequences significantly affects the efficiency of gene editing in the PE system, and depends on the gene locus. Therefore, we investigated the optimization of editing efficiency by combining PBS and RT template sequences of different lengths.

At the cellular level, the effect of gene editing by the FrCas9-PE system was previously tested. In HEK293T cells, the HEK293T-RNF2 gene locus, which is commonly used to assess the efficiency of CRISPR gene editing, was targeted to verify the gene editing function of the FrCas9-PE system. Our current experimental results indicate that FrCas9-PE may have site-specific base editing effects. After that, we will further transform the existing FrCas9-PE, including codon optimization, optimization of the position and number of nuclear localization sequences, reverse transcriptase modification, etc. As a result, a novel FrCas9-PE genome editing system was developed with high base editing efficiency, fewer off-target reactions, and fewer gene editing by-products (indels) (Figure 14).

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Sequence listing

SEQUENCE LISTING

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Lys Ser Ser Leu Leu Tyr Gln Glu Phe Glu Val Leu Asn Glu Leu Asn

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Lys Arg Met Ile Phe Glu Gln Cys Phe Tyr Ser Gly Lys Lys Val Thr

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Gly Lys Lys Leu Gln Leu Phe Leu Arg Ser Val Leu Thr Asn Ser Ser

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Thr Glu Glu Phe Val Leu Thr Gly Ile Asp Lys Asp Phe Lys Ser Ser

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Asn Asp Thr Gln Lys Val Met Ala Glu Gln Ile Ile Glu Trp Ser Thr

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Ser Ile Glu Pro Asp Asp Leu Asp Gly Met Tyr Leu Ser Ala Pro Val

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Arg Arg Met Ile Trp Gln Thr Phe Leu Ile Leu Arg Glu Val Val Asp

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Thr Ile Gly Tyr Ser Pro Lys Lys Ile Phe Met Glu Met Ala Arg Gly

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Glu Gln Glu Lys Lys Arg Thr Ala Ser Arg Lys Lys Gln Leu Ile Asp

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Glu Ser Leu Glu Glu Ala Gln Leu Arg Ser Lys Lys Leu Tyr Leu Tyr

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Lys Ser Gly Asn Leu His Ser Tyr Ser Leu Arg Arg Met Tyr Asp Phe

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Asn Val Gln Arg Gly Asp Gln Thr Ala Trp Val Ala Glu Asn Asp Thr

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Asn Leu Ser Leu Ser Asn Ala Val Glu Leu Cys Leu Pro Ala Lys Glu

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Gln Ala His Ile Arg Met Ile Ser Lys Ile Ala Gly Gly Arg Ser Thr

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Asp Ala Leu Ser Ala Glu Ala Lys Asp Asp Phe Arg Lys Lys Asn Leu

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Arg Leu Tyr Asp Glu Leu Ala Glu Lys His Arg Ser Thr Ile Phe Ser

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Gln Met Gly Tyr Leu Val Val Arg Gly Glu Ser Ile Lys Arg Ile His

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Leu Ser Glu Ile Ser Val Leu Ile Ile Glu Asn Thr Ala Val Ser Leu

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Thr Ala Tyr Leu Val Ser Glu Leu Val Lys Asn Lys Ile Lys Leu Leu

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Phe Cys Asp Glu Lys Arg Ser Pro Leu Ala Glu Val Ser Glu Leu Tyr

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Gly Gly His Asp Ser Ser Asp Met Val Arg Lys Gln Ile Glu Ile Pro

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Ser Asn Gln Phe Ala Val Leu His Asn Phe Asp Cys Pro Asn Gln Glu

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Arg Glu Gly His Ala Ala Lys Val Tyr Phe Asn Ser Leu Phe Gly Lys

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Ser Phe Tyr Arg Ala Ser Glu Cys Ala Leu Asn Ala Ala Leu Asn Tyr

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Gly Tyr Ser Val Leu Leu Ser Ala Val Ser Arg Glu Ile Ala Gly Tyr

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Gly Phe Leu Thr Gln Leu Gly Ile Phe His Asp Asn Cys Asp Asn Lys

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Tyr Asn Leu Ser Cys Asp Leu Met Glu Pro Phe Arg Pro Val Val Asp

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Tyr Leu Val Lys Ser Asn Ile Val Glu Val Phe Glu Lys Glu Gln Lys

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Gln Lys Ile Leu Gln Leu Leu Gln Phe Lys Ile Gln Ile Asn Asp Arg

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Gln Glu Thr Val Gln Asn Ala Ile Ser Ile Phe Val His Ser Val Leu

260 265 270

Asp Tyr Leu Leu Asp Pro Ser Val Tyr Ile Lys Val Pro Arg Ile Asp

275 280 285

Phe Thr Lys Asn Val Val

290

<210> 3

<211> 68

<212> PRT

<213> Faecalibaculum rodentium

<400> 3

Met Met Gln Glu Ser Val Tyr Cys Lys Leu Thr Thr Asn Gln Ser Ser

1 5 10 15

Ala Glu Thr Val Leu Lys Met Val Arg Ala Asn Lys Pro Pro Glu Gly

20 25 30

Leu Ile Gln Thr Leu Ile Ile Thr Glu Lys Gln Phe Ser Lys Met Asp

35 40 45

Phe Ile Leu Gly Gln Pro Asn Ser Asp Val Val Ala Thr Asp Glu Ser

50 55 60

Val Leu Asp Leu

65

<210> 4

<211> 223

<212> PRT

<213> Faecalibaculum rodentium

<400> 4

Met Arg Leu Leu Ile Asp Arg Leu Leu Leu Ser Ala Glu Leu Asn Ile

1 5 10 15

Asp Lys Ala Thr Thr Ile Ile Ile Glu Asn Pro Lys Ala Phe Arg Met

20 25 30

Val Ile Lys Asp Leu Ile Glu Gln Glu Asn Gly Gln Gly Gly Leu Leu

35 40 45

Arg Ile Val Glu Gly Asp Lys Glu Leu Cys Leu Ser Lys Ser Ala Ile

50 55 60

Leu Val Leu Asn Pro Tyr Leu Ala Asp Leu Asn Cys Arg Lys Phe Leu

65 70 75 80

Gln Leu Ala Tyr Ser Glu Leu Gln Ala Met Thr Gly Glu Phe Leu Glu

85 90 95

Asp Gln Ala Val Val Leu Ser Ala Met Thr Gly Tyr Leu Ser Lys Ile

100 105 110

Cys Asp Gln Ser Arg Phe Asp Phe Leu Glu Phe Ser Ala Ile Pro Asp

115 120 125

Trp Ala Ser Val Phe Lys Ala Trp Gly Leu Arg Phe Glu Gln Ala Ile

130 135 140

Pro Gly Leu Leu Pro Ser Leu Ile Gln Tyr Leu Gln Leu Ala Ala Thr

145 150 155 160

Phe Pro Gln Phe Lys Leu Ile Ile Phe Ile Asn Leu Lys Gln Tyr Leu

165 170 175

Leu Pro Glu Glu Gln Phe Glu Leu Phe Lys Met Ala Glu Tyr Leu Gln

180 185 190

Leu Lys Val Leu Leu Val Glu Ser Ala Gln Asn Tyr Lys Ser Asp Arg

195 200 205

Glu Asp Leu Ile Ile Ile Asp Lys Asp Leu Cys Glu Ile Gln Ser

210 215 220

<210> 5

<211> 20

<212>RNA

<213> Faecalibaculum rodentium

<400> 5

guuugagugu cuuguuaauu 20

<210> 6

<211> 36

<212> DNA

<213> Faecalibaculum rodentium

<400> 6

gtttgagtgt cttgttaatt cggaagtatt tcaaac 36

<210> 7

<211> 216

<212> DNA

<213> Faecalibaculum rodentium

<400> 7

60

120

180

gtttgagtgt cttgttaatt cggaagtatt tcaaac 216

<210> 8

<211> 71

<212>RNA

<213> Faecalibaculum rodentium

<400> 8

aauuaacaag augaguucaa aucaggcucc uagagagauc cgaacuuacc uucauggcgg 60

gcauugugcc c 71

<210> 9

<211> 95

<212>RNA

<213> Faecalibaculum rodentium

<400> 9

guuugagugu cuuguuaauu gaaaaauuaa caagaugagu ucaaaucagg cuccuagaga 60

gauccgaacu uaccuucaug gcgggcauug ugccc 95

<210> 10

<211> 91

<212>RNA

<213> Faecalibaculum rodentium

<400> 10

guuugagugu cuuguuaaga aauuaacaag augaguucaa aucaggcucc uagagagauc 60

cgaacuuacc uucauggcgg gcauugugcc c 91

<210> 11

<211> 89

<212>RNA

<213> Faecalibaculum rodentium

<400> 11

guuugagugu cuuguuagaa auaacaagau gaguucaaau caggcuccua gagagauccg 60

aacuuaccuu cauggcgggc auugugccc 89

<210> 12

<211> 87

<212>RNA

<213> Faecalibaculum rodentium

<400> 12

guuugagugu cuuguugaaa aacaagauga guucaaauca ggcuccuaga gagauccgaa 60

cuuaccuuca uggcgggcau ugugccc 87

<210> 13

<211> 85

<212>RNA

<213> Faecalibaculum rodentium

<400> 13

guuugagugu cuugugaaaa caagaugagu ucaaaucagg cuccuagaga gauccgaacu 60

uaccuucaug gcgggcauug ugccc 85

<210> 14

<211> 83

<212>RNA

<213> Faecalibaculum rodentium

<400> 14

guuugagugu cuuggaaaca agaugaguuc aaaucaggcu ccuagagaga uccgaacuua 60

ccuucauggc gggcauugug ccc 83

<210> 15

<211> 79

<212>RNA

<213> Faecalibaculum rodentium

<400> 15

guuugagugu cugaaaagau gaguucaaau caggcuccua gagagauccg aacuuaccuu 60

cauggcggc auugugccc 79

<210> 16

<211> 75

<212>RNA

<213> Faecalibaculum rodentium

<400> 16

guuugagugu gaaaaugagu ucaaaucagg cuccuagaga gauccgaacu uaccuucaug 60

gcgggcauug ugccc 75

<210> 17

<211> 71

<212>RNA

<213> Faecalibaculum rodentium

<400> 17

guuugaguga aagaguucaa aucaggcucc uagagagauc cgaacuuacc uucauggcgg 60

gcauugugcc c 71

<210> 18

<211> 69

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 18

ctacctgcat accgttatta acatatgaca actcaattaa acgccacatc catcggcgct 60

ttggtcggc 69

<210> 19

<211> 62

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 19

atgttaccac tggattgagg ataccgttat taacatatga caactcaatt aaactctggt 60

ac62

<210> 20

<211> 69

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 20

tcagtttata tgagttacaa cgaacaccgt ttaattgagt tgtcatatgt taataacggt 60

attcaggta 69

<210> 21

<211> 69

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 21

ctacctacat accgttatta acatatgaca actcaattaa acgtcagcac ctgggacccc 60

gccaccgtg 69

<210> 22

<211> 34

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 22

gtttaattga gttgtcatat gttaataacg gtat 34

<210> 23

<211> 26

<212> DNA

<213> Artificial Sequence

<221> misc_feature

<222> (22)..(26)

<223> n is a, c, g, or t;r is a or g;w is a or t

<400> 23

tgttaccact ggattgaggt cnnrwr 26

<210> 24

<211> 26

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 24

tgttaccact ggattgaggt ctggta 26

<210> 25

<211> 26

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 25

aagaaccact ggattgaggt ctggct 26

<210> 26

<211> 26

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 26

gggaaccact ggattaaggt ccagat 26

<210> 27

<211> 26

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 27

agttaccaat ggattgagat ggggag 26

<210> 28

<211> 26

<212> DNA

<213> Artificial Sequence

<221> misc_feature

<222> (22)..(26)

<223> n is a, c, g, or t;r is a or g;w is a or t

<400> 28

aaaaaggaag gagttctttg tnnrwr 26

<210> 29

<211> 26

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 29

aaaaaggaag gagttctttg taggta 26

<210> 30

<211> 26

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 30

taaaaggaag gtgttctttg tggggt 26

<210> 31

<211> 26

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 31

gaaaaggaag gtgctctttg tgggtg 26

<210> 32

<211> 26

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 32

ggaaaaggtg gaattctttg ttggta 26

<210> 33

<211> 17

<212> DNA

<213> Artificial Sequence

<223> Synthetic

<400> 33

cgaacacctc aggtaat 17

<---

Claims (37)

1. Type II CRISPR/Cas9 genome editing system, characterized in that the system contains the Cas9 protein, accessory proteins, crRNA (CRISPR RNA) and tracrRNA (transactivated CRISPR RNA) in the functional form of the RNP complex (ribonucleoprotein complex) of the Cas9 protein and guide RNA, formed by hybridization of crRNA with tracrRNA; wherein the Cas9 protein is a DNA endonuclease and has an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having the same function of the amino acid sequence as shown in SEQ ID NO: 1 and having at least 80% , 85%, 90%, 95%, 98% or 99% homology with the amino acid sequence as shown in SEQ ID NO: 1; where CRISPR RNA is generated by transcription of a CRISPR array and has an RNA sequence as shown in SEQ ID NO: 5, or an RNA sequence having the same function as an RNA sequence as shown in SEQ ID NO: 5 and having at least 80%, 85%, 90%, 95%, 98% or 99% homology to the nucleic acid sequence as shown in SEQ ID NO: 5; And where the transactivated CRISPR RNA contains a sequence complementary to the CRISPR RNA direct repeat sequence and the transactivated CRISPR RNA has a nucleic acid sequence as shown in SEQ ID NO: 8 or a nucleic acid sequence having the same function as a nucleic acid sequence as shown in SEQ ID NO: 8, and having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% homology with the nucleic acid sequence as shown in SEQ ID NO: 8. 2. Type II CRISPR/Cas9 genome editing system according to claim 1, characterized in that the Cas9 protein cleaves double-stranded DNA complementary to crRNA above the PAM sequence using a nuclease domain, while the nuclease domain is selected from the HNH-like nuclease domain, RuvC- a similar nuclease domain; or a combination thereof. 3. The type II CRISPR/Cas9 genome editing system according to claim 1, wherein the accessory proteins include the Cas1 accessory protein, the Cas2 accessory protein, and the Csn2 accessory protein; wherein the Cas1 accessory protein has an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having the same function of the amino acid sequence as shown in SEQ ID NO: 2 and having at least 80%, 85%, 90 %, 95%, 98% or 99% homology to the amino acid sequence as shown in SEQ ID NO: 2; wherein the Cas2 accessory protein has an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having the same function of the amino acid sequence as shown in SEQ ID NO: 3 and having at least 80%, 85%, 90 %, 95%, 98% or 99% homology to the amino acid sequence as shown in SEQ ID NO: 3; wherein the Csn2 accessory protein has an amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having the same function of the amino acid sequence as shown in SEQ ID NO: 4 and having at least 80%, 85%, 90 %, 95%, 98% or 99% homology to the amino acid sequence as shown in SEQ ID NO: 4. 4. Type II CRISPR/Cas9 genome editing system according to claim 1, characterized in that the CRISPR array contains a direct repeat sequence and a spacer sequence, where the direct repeat sequence has a nucleic acid sequence as shown in SEQ ID NO: 6, or a nucleic acid sequence acid having the same function of the nucleic acid sequence as shown in SEQ ID NO: 6 and having at least 80%, 85%, 90%, 95%, 98% or 99% homology with the nucleic acid sequence as shown in SEQ ID NO: 6; and where the spacer sequence has a nucleic acid sequence as shown in SEQ ID NO: 7, or a nucleic acid sequence having the same function as a nucleic acid sequence as shown in SEQ ID NO: 7, and having at least 80%, 85% , 90%, 95%, 98% or 99% homology to the nucleic acid sequence as shown in SEQ ID NO: 7. 5. Type II CRISPR/Cas9 genome editing system according to claim 1, characterized in that the guide RNA formed by hybridization of crRNA with tracrRNA has a framework consisting of a sequence of 7–24 nucleotides of a crRNA direct repeat sequence and a tracrRNA sequence; preferably the two parts are fused with a "GAAA", "TGAA" or "AAAC" linker to form an sgRNA backbone; preferably an sgRNA framework formed by fusing a 20 nt crRNA direct repeat sequence and a full-length tracrRNA sequence with "GAAA" is shown in SEQ ID NO: 9, and on this basis, preferably a highly efficient variant of the sgRNA framework is an sgRNA backbone formed by fusing the first 18-14 nucleotides of a crRNA direct repeat sequence and the last 69-65 nucleotides of tracrRNA with a linker sequence that is GAAA, preferably selected from the following five scaffolds: (1) a 91 nucleotide long sgRNA backbone that contains 18 nucleotides of the direct repeat sequence and 69 nucleotides of tracrRNA as shown in SEQ ID NO: 10; (2) an 89 nt sgRNA backbone that contains 17 nt of the direct repeat sequence and 68 nt of tracrRNA as shown in SEQ ID NO: 11; (3) an 87 nt sgRNA backbone that contains 16 nt of direct repeat sequence and 67 nt of tracrRNA as shown in SEQ ID NO: 12; (4) an 85 nt sgRNA backbone that contains 15 nt of direct repeat sequence and 66 nt of tracrRNA as shown in SEQ ID NO: 13; (5) an 83 nucleotide long sgRNA backbone that contains 14 nucleotides of the direct repeat sequence and 65 nucleotides of tracrRNA as shown in SEQ ID NO: 14; optionally, the sgRNA backbone has a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homology to any of SEQ ID NOs: 9-14. 6. Type II CRISPR/Cas9 genome editing system according to claim 1, characterized in that the type II CRISPR/Cas9 genome editing system is derived from Faecalibaculum rodentium , and in which specific DNA is bound or cleaved during the genome editing process, by recognizing complementary guide pairing. RNA and specific DNA targets, wherein the length of the paired binding portion of the guide RNA and specific DNA target is 14 to 30 base pairs; Wherein the specific DNA is prokaryotic or eukaryotic DNA. 7. Type II CRISPR/Cas9 genome editing system according to claim 6, characterized in that the length of the paired binding portion of the guide RNA and the specific DNA target is 21 base pairs, 22 base pairs, or 23 base pairs, and the RNP complex is very sensitive to mismatch of 14 base pairs close to the motif adjacent to the protospacer, and 14 base pairs are the seed region. 8. The type II CRISPR/Cas9 genome editing system of claim 7, wherein the motif adjacent to the protospacer required for DNA binding or cleavage function is 5'-NNTA-3' towards the lower end of the guide RNA recognition sequence/ sgRNA where N is A, T, C or G. 9. Use of the type II CRISPR/Cas9 genome editing system according to any one of claims 1 to 8 for DNA editing. 10. Use according to claim 9, wherein the DNA is prokaryotic or eukaryotic DNA. 11. The use of the CRISPR/Cas9 type II genome editing system according to any one of paragraphs. 1-8 for DNA binding. 12. Use according to claim 11, wherein the DNA is prokaryotic or eukaryotic DNA. 13. The use of the CRISPR/Cas9 type II genome editing system according to any one of paragraphs. 1-8 for CRISPR activation. 14. The use of the CRISPR/Cas9 type II genome editing system according to any one of paragraphs. 1-8 for CRISPR interference. 15. The use of the CRISPR/Cas9 type II genome editing system according to any one of paragraphs. 1-8 to get a niqaza. 16. Use according to claim 15, wherein the niqase is produced in a prokaryote or eukaryote. 17. The use of the CRISPR/Cas9 type II genome editing system according to any one of paragraphs. 1-8 to get a dead Cas9. 18. Use according to claim 17, wherein the dead Cas9 is obtained in a prokaryote or eukaryote. 19. The use of the CRISPR/Cas9 type II genome editing system according to any one of paragraphs. 1-8 to get the reason editor. 20. Use according to claim 19, wherein the base editor is produced in a prokaryote or eukaryote. 21. The use of the CRISPR/Cas9 type II genome editing system according to any one of paragraphs. 1-8 to get the chief editor. 22. Use according to claim 21, where the chief editor is obtained in a prokaryote or eukaryote.

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