JP2023121643A - Type ii crispr/cas9 genome editing system and the application thereof - Google Patents

Abstract

To provide a Type II CRISPR/Cas9 genome editing system.SOLUTION: Provided herein is a Type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, comprising a Cas9 protein, an accessory protein, a CRISPR RNA and a trans-activated CRISPR RNA; where the Cas9 protein is a DNA endonuclease, and the Cas9 protein has a specific amino acid sequence, or an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the specific amino acid sequence.SELECTED DRAWING: None

Description

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

Gene editing technologies allow for the remodeling of DNA sequence locations, such as the first generation of gene editing tools zinc finger nucleases (ZFNs) and the second generation of gene editing tools, zinc finger nucleases (ZFNs). Gene-editing tools, transcription activator-like effector nucleases (TALENs), can all be used to modify target genomes, but these methods are difficult to design, difficult to produce, and costly. And there is a problem that versatility is not good.
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR-associated protein) system is an innate immune system derived from archaea and bacteria. It is a system and a third generation gene editing tool. Unlike conventional gene-editing tools (protein-DNA recognition), this method utilizes the principle of nucleobase pair complementarity to recognize target DNA sequences and Cas effector proteins are site-specific. It is highly adaptable, simple in design, low in cost and high in efficiency. Cas proteins contain a variety of different effector domains that play roles in a variety of activities such as nucleic acid recognition, structural stabilization of complexes, and hydrolysis of phosphodiester bonds in DNA. Among them, the type II CRISPR/Cas9 system (Streptococcus pyogene Cas, SpCas9) derived from Streptococcus pyogenes is currently the most widely used CRISPR/Cas system due to its high cleavage efficiency. This system recognizes and cleaves the protospacer adjacent motif (PAM) sequence "NGG" in the target polynucleotide, leaving a single blunt or sticky end, affecting gene editing.
The vast and diverse metagenome harbors uncultivated or undiscovered microbes, the potential for many undiscovered CRISPR/Cas9 systems, and the prokaryotic and eukaryotic evolution of these systems. It is necessary to confirm the activity in organisms and in vitro environments.
In 2015, Dr. Byoung-Chan Kim's team isolated a new anaerobic strain ALO17 from the feces of laboratory mice C57BL/6J, and by phylogenetic analysis of the sequence of the prokaryotic 16S rRNA gene, the strain was named Holdemanellabiformis DSM 3989T. , Faecalicoccus pleomorphus ATCC 29734T, Faecalitaleacylindroides ATCC 27803T, and Allobaculumstercoricanis DSM 13633T (sequence identities of 87.4%, 87.3% and 8%, respectively). 6.9%, and 86 .9%). Based on a large body of taxonomic evidence, this species has been assigned a new genus in the family Erysipelothricaceae, and Faecalibaculum rodentium gen. nov. , sp. nov. was named. Over the next five years, scientists around the world have studied this strain in two areas: gut microbiome and high-fat diet, gut microbiome and tumorigenesis, but nothing has yet been reported on gene editing. do not have.

The present invention overcomes the deficiencies of the prior art, has new physicochemical properties, and is capable of producing multiple different PAM (protospacer adjacent motifs) including NGTA and NNTA (N represents A, C, G or T). It is an object of the present invention to provide a type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium that is capable of recognizing Flanking Motif) sequences.
In order to achieve the above objects, the technical means adopted in the present invention are:
A type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium, comprising Cas9 protein, accessory protein, crRNA (i.e. CRISPR RNA) and tracrRNA (i.e. transactivating CRISPR RNA), wherein Cas9 functions in the form of an RNP complex (i.e., a ribonucleoprotein complex) of a protein and a guide RNA formed by binding tracrRNA to crRNA,
The Cas9 protein is a DNA endonuclease, and the Cas9 protein is the amino acid sequence shown in SEQ ID NO: 1, or at least 80%, 85%, 90%, 95%, 98% of the amino acid sequence shown in SEQ ID NO: 1 %, or 99% homology.
Said Cas9 protein cleaves double-stranded DNA complementary to crRNA upstream of the PAM sequence with a nuclease domain, said nuclease domain being an HNH-like nuclease domain and/or a RuvC-like nuclease domain.
In some embodiments, the "ribonucleoprotein complex" is preferably the "FrCas9 protein complex" in the present invention.
The mutational status of some key amino acid positions of specific Cas9 proteins is described. Mutation of E at amino acid position 796 to A results in a Nickase nuclease, mutation of N at amino acid position 902 to A results in a Nickase nuclease, H at amino acid position 1010 to A. Mutation results in a nickase nuclease, mutation of D at amino acid position 1013 to A results in nickase nuclease, and mutation of E at amino acid position 796 and D at amino acid position 1013 to A at the same time. It results in a Cas9 nuclease that is incapable of cleaving but retains binding ability (ie, inactive Cas9 (dead Cas9)).
The accessory proteins include Cas1 accessory protein, Cas2 accessory protein and Csn2 accessory protein,
The Cas1 accessory protein is the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence set forth in SEQ ID NO: 2 has an array,
The Cas2 accessory protein is the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence set forth in SEQ ID NO: 3 has an array,
The Csn2 accessory protein is the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence set forth in SEQ ID NO: 4 has an array.
The CRISPR RNA is generated by transcription from a CRISPR array, and the CRISPR RNA is at least 80%, 85%, 90%, 95% of the RNA sequence set forth in SEQ ID NO:5 or the nucleic acid sequence set forth in SEQ ID NO:5. , 98% or 99% homology, wherein the CRISPR array comprises a direct repeat sequence and a spacer sequence, wherein the direct repeat sequence is shown in SEQ ID NO: 6 a nucleic acid sequence or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the nucleic acid sequence set forth in SEQ ID NO: 6, wherein the spacer sequence comprises 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 shown in SEQ ID NO:7.
The transactivating CRISPR RNA contains a sequence complementary to a direct repeat sequence of CRISPR RNA, and the transactivating CRISPR RNA is the nucleic acid sequence shown in SEQ ID NO: 8, or the nucleic acid shown in SEQ ID NO: 8 Have a nucleic acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homologous to the sequence.
The backbone of the guide RNA, which is crRNA bound to tracrRNA, is composed of 7-24 nt of the crRNA direct repeat sequence and the corresponding length of the tracrRNA sequence, and these two parts are designated as "GAAA", "TGAA", can be fused via a linker such as "AAAC" to form an sgRNA backbone, preferably a sgRNA backbone formed by fusing a 20 nt direct repeat sequence and a 71 nt tracrRNA sequence via a "GAAA" is shown in SEQ ID NO: 9, and further preferred variants based on the sgRNA backbone are:
an sgRNA backbone formed by fusing the first 18-14 nt of the direct repeat sequence of the crRNA and the last 69-65 nt of the tracrRNA through a linker sequence (such as "GAAA"), which further comprises
(a) a backbone represented by SEQ ID NO: 10, which has a length of 91 nt and contains a direct repeat sequence of 18 nt and a tracrRNA of 69 nt;
(b) an sgRNA backbone having a length of 89 nt, containing a direct repeat sequence of 17 nt and a tracrRNA of 68 nt, and represented by SEQ ID NO: 11;
(c) an sgRNA backbone of 87 nt in length, containing a 16 nt direct repeat sequence and 67 nt of tracrRNA, and represented by SEQ ID NO: 12;
(d) an sgRNA backbone having a length of 85 nt, containing a direct repeat sequence of 15 nt and a tracrRNA of 66 nt, and represented by SEQ ID NO: 13;
(e) a backbone represented by SEQ ID NO: 14, which has a length of 83 nt and contains a direct repeat sequence of 14 nt and a tracrRNA of 65 nt;
A skeleton selected from the group consisting of
The sgRNA backbone may have a nucleic acid sequence with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% homology to any one of SEQ ID NOs:9-14.
The type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium binds or cleaves specific DNA in a biological process (e.g., gene editing). The range of the length of the binding portion that is realized by recognizing the guide RNA by complementing and pairing with the target site of the specific DNA, and the pairing portion of the guide RNA and the target site of the specific DNA is , 14-30 bp (preferably 20-23 bp), and the specific DNA is prokaryotic or eukaryotic DNA.
The length of the paired binding portion of the guide RNA and the specific DNA target site is 21 bp, 22 bp or 23 bp, wherein the RNP protein complex is adjacent to a protospacer adjacent motif (PAM). It is very sensitive to 14 bp base mismatches, said 14 bp being the seed region.
The protospacer-adjacent motif sequence required for the DNA binding or cleaving function is 5'-NNTA-3' downstream of the guide RNA/sgRNA recognition sequence.
The present invention further provides a CRISPR/Cas9 gene editing system for editing or joining DNA, activating CRISPR or interfering with CRISPR, wherein the CRISPR/Cas9 gene editing system comprises the type II CRISPR/Cas9 gene Editing system. Preferably, said DNA is prokaryotic or eukaryotic DNA. Preferably, said type II CRISPR/Cas9 gene editing system is derived from Faecalibaculum rodentium.
In another aspect, the present invention also provides a CRISPR/Cas9 gene editing system for the production of Nickase, Inactive Cas9, Base Editor or Prime Editor, wherein said CRISPR/Cas9 The gene editing system is the Type II CRISPR/Cas9 gene editing system described above. Preferably, the process of producing the nickase, inactive Cas9, Base Editor or Prime Editor is performed in a prokaryote or eukaryote.
The beneficial effects of the present invention compared with the prior art are as follows.
(1) The present invention discovers a type II CRSIPR/Cas9 gene editing system in Faecalibaculum rodentium through bioinformatics analysis, and the gene editing system can be applied to edit prokaryotic or eukaryotic genes, gene editing tools Offer new options in the box.
(2) The type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium provided by the present invention has new physicochemical properties, can recognize a plurality of different PAM sequences, and the gene editing system A specific sequence of PAM that is recognized is 5'-NNTA-3' downstream of the guide RNA/sgRNA recognition sequence.
(3) The type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium provided by the present invention has a higher cutting efficiency at the same DNA site than the most commonly used SpCas9, and the most commonly used off-target rate It is a safer and more effective gene-editing tool than SpCas9.
(4) In the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium provided by the present invention, the PAM recognized by the gene editing system has palindrome characteristics, and the target site is on the genome distributed "back-to-back" in , with a higher density than SpCas9.
(5) The type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium provided by the present invention has high flexibility and can be remodeled into a base editor (Base Editor) and a prime editor (Prime Editor), It is a gene editing tool that can be applied in different scenarios.

1 is a schematic diagram of the type II CRISPR/Cas9 gene editing system of Faecalibaculum rodentium according to the present invention. FIG. Fig. 2 is a structural schematic diagram of the Cas9 protein of the type II CRISPR/Cas9 gene editing system of Faecalibaculum rodentium according to the present invention; FIG. 2 is a schematic diagram of an optimal sgRNA backbone and a predicted RNA secondary structure of a guide RNA molecule recognized by the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 3A shows the full length of the 95nt gRNA. FIG. 3B shows a gRNA with a backbone length of 91 nt, 18 nt direct repeat plus 69 nt tracrRNA. FIG. 3C shows an sgRNA with a backbone length of 89 nt, 17 nt direct repeat plus 68 nt tracrRNA. FIG. 3D shows a gRNA with a backbone length of 87 nt, 16 nt direct repeat plus 67 nt tracrRNA. FIG. 3E shows an sgRNA with a backbone length of 85 nt, 15 nt direct repeat plus 66 nt tracrRNA. FIG. 3F shows an sgRNA with a backbone length of 83 nt, 14 nt direct repeat plus 65 nt tracrRNA. FIG. 2 is a schematic representation of the prokaryotic PAM sequence of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the invention. FIG. 2 is a schematic diagram of a prokaryotic interference experiment of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 2 is a schematic representation of eukaryotic cleavage of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 6A is an ODN-PCR gel electropherogram. FIG. 6B is a peak diagram from Sanger sequencing. FIG. 2 is a schematic diagram of the optimal length of sgRNA for the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 7A shows cleavage efficiency of different sgRNA lengths at the HEK293 SITE2-T2 site. Figure 7B shows the cleavage efficiency of different sgRNA lengths at the DNMT1-T3 site. FIG. 7C shows the cleavage efficiency of different sgRNA lengths at the RNF2-T6 site. Fig. 2 shows the optimal length of the recognition sequence of sgRNA of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 3 is a schematic diagram showing a comparison of the target efficiency and off-target in GUIDE-seq of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention and the target efficiency and off-target in SpCas9 in GUIDE-seq. FrCas9-BE base editor experimental results. Here, FIG. 10A is a principle diagram and results for confirming the form of FrCas9 nickase by ODN breakpoint PCR. FIG. 10B is the edit window of nFrCas9-BE4Gram. FIG. 10C is the edit window of nFrCas9-ABE7.10. FIG. 10D is a Venn diagram of the unique pathogenic mutations that can be corrected with the SpCas9 and FrCas9 base editors in the ClinVar database. FIG. 10E shows C>T editing efficiency of FrCas9-BE4Gam using two “back-to-back” sgRNAs simultaneously. FIG. 2 is a distribution map of target sites of FrCas9 and SpCas9 in the human genome. Here, Figure 11A shows the distribution of 5'-GG-3' (representing SpCas9 PAM) in the GRCh38 human genome. Figure 11B shows the distribution of 5'-TA-3' (representing FrCas9 PAM) in the GRCh38 human genome. FIG. 4 shows the application of FrCas9 specifically targeting the TATA box to CRISPR interference and CRISPR activation. Here, FIG. 12A is a schematic diagram of the TATA box of the ABCA1 gene, which is the target of FrCas9. FIG. 12B shows CRISPR interference in FrCas9 and SpCas9 by targeting the TATA box and CRISPR activation in FrCas9 and SpCas9 by targeting the TATA box. FIG. 12C shows CRISPR activation in FrCas9 and SpCas9 by targeting the TATA box. Schematic diagram of a gene editing system for prime editing. FIG. 4 shows validation of the gene-editing action of the FrCas9-PE prime editing system by Sanger sequencing. The measured sequence is CGAACACCTCAGGTAAT (SEQ ID NO:33).

In order to better describe the purpose, technical solution and advantages of the present invention, the present invention is further described below based on specific examples.
The type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium contains Cas9 protein, accessory proteins, CRISPR RNA and transactivating CRISPR RNA, and as shown in FIG. 1, the Cas9 protein is a DNA endonuclease. wherein the Cas9 protein has at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 1 It has an amino acid sequence.
As shown in Figure 2, as a preferred embodiment of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention, the Cas9 protein is complemented with crRNA upstream of the PAM sequence by various nuclease domains. The various nuclease domains are HNH-like nuclease domains or RuvC-like nuclease domains.
The Cas9 protein (Faecalibaculum rodentium Cas9) according to the present invention, sometimes simply called FrCas9 protein, contains 1372 amino acids (SEQ ID NO: 1) and is a multidomain and multifunctional DNA endonuclease. The Cas9 protein efficiently cleaves double-stranded DNA complementary to sgRNA upstream of the PAM by means of various nuclease domains. For example, the HNH-like nuclease domain cleaves the DNA strand complementary to the sgRNA sequence, and the RuvC-like nuclease domain cleaves the non-complementary DNA strand. Here, mutation of E at amino acid position 796 to A results in a nickase nuclease, mutation of N at amino acid position 902 to A results in a nickase nuclease, and H at amino acid position 1010 to A. Mutation of D at amino acid position 1013 to A results in nickase nuclease, E at amino acid position 796 and D at amino acid position 1013 are simultaneously mutated to A This results in a Cas9 nuclease that is incapable of cutting but retains binding ability.
As a preferred embodiment of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention, said accessory proteins comprise a Cas1 accessory protein, a Cas2 accessory protein and a Csn2 accessory protein, wherein said Cas1 accessory protein has the sequence Having an amino acid sequence represented by number 2 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence represented by SEQ ID NO: 2, said Cas2 The accessory protein has the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:3. wherein the Csn2 accessory protein has the amino acid sequence set forth in SEQ ID NO:4 or at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:4 has an amino acid sequence with
The accessory proteins Cas1, Cas2, Csn2 according to the invention are involved in foreign gene capture and maturation of crRNA.
As a preferred embodiment of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention, the CRISPR RNA is produced by being transcribed from a CRISPR array, and the CRISPR RNA is the RNA shown in SEQ ID NO: 5 wherein said CRISPR array comprises direct repeat sequences and spacer sequences, said direct repeat sequences being shown in SEQ ID NO:6 and said spacer sequences being shown in SEQ ID NO:7.
The CRISPR RNAs (crRNAs) described in the present invention exploit base-pair complementarity to induce Cas proteins to recognize invading foreign genomes. Bacteria, when exposed to bacteriophages, viruses, etc., provide a genetic record of infection by incorporating short pieces of foreign DNA as new spacers between CRISPR repeat spacer sequences in the host chromosome; When a foreign gene re-invades the organism, a crRNA precursor (pre-crRNA) is generated by being transcribed from the CRISPR array, and its 5′ end is complementary to the sequence of the gene that has invaded from outside, and is 30 bp in length. A spacer sequence has a repeat sequence 36 bp in length at the 3' end.
As a preferred embodiment of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention, said transactivating CRISPR RNA contains a sequence complementary to a CRISPR RNA direct repeat sequence, and said transactivating A type CRISPR RNA is shown in SEQ ID NO:8.
Trans-activating crRNAs (tracrRNAs) according to the present invention are RNAs that do not encode proteins and are involved in maturation of crRNAs and formation of sgRNAs. The pre-crRNA cleaves 0-16 nt upstream of the spacer sequence and 12-29 nt downstream of the repeat sequence by the action of tracrRNA and Cas9 nuclease to form mature crRNA, which further binds to tracrRNA to form a tracrRNA-crRNA complex. form the body. The complex is 14-30 bp in length and contains a portion that recognizes a sequence of foreign DNA. By adding a tetraloop of four bases, such as a "GAAA", "TGAA" or "AAAC" sequence, between the crRNA downstream and the tracrRNA upstream, the tracrRNA and crRNA are linked to form an sgRNA. can be formed. Such sgRNAs contain two bulge structures and three duplex structures upstream, and one stem loop structure downstream, and also recognize sequences of foreign DNA. It can be divided into partial and skeletal parts.
By adjusting the length of the portion of the sgRNA that recognizes the foreign DNA sequence and the length of the tracrRNA, the cleavage action of the endonuclease can be further optimized. From previous experiments of the present invention, the optimal length of the portion of the sgRNA that recognizes the foreign DNA sequence is 21 bp, 22 bp or 23 bp (FIG. 8), and the optimal length of the backbone portion is shown in FIGS. 7, 91 nt (18 nt crRNA direct repeat + 69 nt tracrRNA), 89 nt (17 nt crRNA direct repeat + 68 nt tracrRNA), 87 nt (16 nt crRNA direct repeat + 67 nt tracrRNA), 85 nt (15 nt crRNA It was proved that there are five types of 83 nt (direct repeat sequence + 66 nt tracrRNA) and 83 nt (14 nt crRNA direct repeat sequence + 65 nt tracrRNA).
In a preferred embodiment of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention, said type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium binds DNA in a biological process or disconnect.
In a preferred embodiment of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention, said DNA is prokaryotic or eukaryotic DNA.
In some embodiments, the FrCas9 of the present invention can recognize multiple types of protospacer adjacent motifs (PAMs) flanking the downstream side of the target sequence to cause DNA double-stranded molecular breaks (DSBs). can. Recognition of the target sequence by the FrCas9 protein requires two key elements: the nucleotides complementary to the crRNA spacer sequence and the Protospacer Adjacent Motif (PAM) flanking the complementary sequence. As shown in Figure 4, depletion experiments revealed that FrCas9 has a cleaving effect in the prokaryotic system, with positions 3 and 4 of the PAM sequence recognized by this newly discovered type II CRISPR/Cas9 system It was rudimentary verified to be TA. Furthermore, as shown in Figure 5, interference experiments demonstrated that the PAM downstream of the target sequence recognized by FrCas9 was 5'-NNTA-3'. As shown in FIG. 6, all of the above PAMs were verified in eukaryotic experiments. By artificially designing spacer sequences in crRNA, such CRISPR-Cas9 systems can target almost any DNA sequence of interest in the genome, generating site-specific blunt ends. produces a double-strand break (DSB). By repairing the DSB with non-homologous end joining, small random insertions/deletions (indels) at the break site are generated to inactivate the gene of interest. Alternatively, high-fidelity homologous recombination repair can be used to make precise genomic modifications at DSB sites using homologous recombination repair templates.
Many human genetic diseases are due to single nucleotide mutations and cannot be cured by traditional methods. Base editors are one of the newest and most effective ways to achieve precise gene editing. It features a nicking version of the CRISPR-Cas protein that locates specific target DNA and uses DNA deaminase to modify and modify such target DNA without causing double-stranded DNA breaks. Mutate and correct for the diseased base. In some embodiments, the E796A nFrCas9-BEGam cytosine base editor combines E796A nFrCas9 with the optimized fourth generation cytidine base editor BE4Gam to mutate base pairs C:G to T:A in DNA ( E796A nFrCas9-BABE7.10 adenine base editor (ABE) that combines E796A nFrCas9 with the seventh generation adenine base editor ABE7.10 to mutate the base pair A:T to G:C. ) can be constructed.
In some embodiments, the specific PAM of FrCas9 of the present invention is NNTA and has a target site specific to the TATA box (one of the elements that make up the eukaryotic promoter), so FrCas9 is specific By specifically targeting the TATA box, unique CRISPR interference (CRISPRi)/CRISPR activation (CRISPRa) effects can be exerted. Here, CRISPRi either utilizes active FrCas9 to directly target cleave the TATA box to destroy it, or dFrCas9 without cleavage activity (i.e., inactive Cas9) directly binds to the TATA box. It can be realized by letting CRISPRa can be achieved by, but not limited to, dFrCas9-VP64 targeting the TATA box site.
Prime Editing (PE) is an entirely new precision gene editing tool. This technology enables free substitution of single bases and precise insertions and deletions of multiple bases, greatly reducing harmful by-products produced by indels in the process of gene editing, and improving editing accuracy. can be greatly improved and is widely considered to be a significant advance in gene editing. In some embodiments, the FrCas9 of the present invention can be fused with reverse transcriptase and the corresponding pegRNA can be designed based on its PAM sequence to construct the FrCas9-PE gene editing system. Specifically, two sequences are added to the 3' end sequence of pegRNA. The first sequence is the primer binding site (PBS) and can complement the ends of the cleaved target DNA strand to initiate the reverse transcription process, and the second sequence is the reverse transcription template (RT templates) and have targeted point mutations or indel mutations to achieve precise gene editing.
It is a further object of the present invention to provide the use of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium in prokaryotic or eukaryotic gene editing, CRISPR activation or CRISPR interference.
As a preferred embodiment of the use according to the invention, the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium is used to join or cleave DNA at the DNA level.
Example 1
In this example, the RNA secondary structure of the guide RNA molecule recognized by the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention is predicted, and the RNA transcribed from the tracrRNA and the repetitive sequence is predicted. By simulating the binding process, the RNA structure after binding of both was predicted, and the obtained RNA secondary structure is shown in FIG.
(1) Materials: predicted tracrRNA and repeat sequences, and predicted anti-repeat sequences.
(2) Software: NUPACK (http://www.nupack.org/partition/new)
(3) Prediction method: NUPACK, an online application, simulates the in vitro interaction process of 1 µl of each of the two RNAs at 37°C, and predicts the secondary structure of the resulting RNA composition. An RNA secondary structure as shown in 2 was obtained.
As can be seen from FIG. 3, the pre-crRNA is excised by tracrRNA and Cas9 nuclease from 0-16 nt upstream of the spacer sequence and 12-29 nt downstream of the repeat sequence to form the mature crRNA and fuse with the tracrRNA. A tracrRNA-crRNA complex was formed. This complex contained a sequence of foreign DNA complementary to the spacer sequence, with a sequence length of 14-30 bp. Adding a 4-nt tetraloop between the crRNA downstream and the tracrRNA upstream connects them to form two bulge and three double helix structures upstream and three stem-loop structures downstream. can form an sgRNA comprising
Example 2
In this example, the protospacer adjacent motif (PAM) recognized in the prokaryotic system by the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention was found to be 5'-NNTA-3'. .
(1) Materials: genes related to the CRISPR/Cas9 gene editing system predicted by the above examples.
(2) Verification method: In this example, a prokaryotic verification system was constructed for the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention, the cleavage effect was verified, and the recognized PAM sequence was preliminarily searched by second generation sequencing technology and the results are shown in FIG.
The detailed flow is as follows.
(a) Type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention, comprising endonuclease Cas9, accessory proteins Cas1, Cas2, Csn2, CRISPR array and non-coding RNA tracrRNA ) was inserted into the pACYC184 vector, E. coli codon-optimized for the Cas9 protein, the CRISPR array was added with the native spacer sequence and the spacer sequence in the library, and the Cas9 protein, a strong heterologous promoter upstream of the CRISPR array. (heterologous promoter) J23119 was added to construct a prokaryotic expression pACYC184-FrCas9 plasmid.
(b) adding 7 random bases (total of 16384 inserts) to the 3′ end of the spacer sequence of the library and cleaving the multiple cloning site (MCS) of the pUC19 vector with two enzymes, EcoRI and NcoI sites; Sites were selected and the library cloned into a vector to construct the target-library plasmid.
(c) Both the pACYC184 plasmid containing pACYC184-FrCas9 or the empty vector and the target-library plasmid were introduced into E. coli DH5a by the electrotransformation method, revived at 25°C for 2 hours, and then treated with ampicillin sodium (100 µg/ml). and chloramphenicol (34 μg/ml), and incubated at 25° C. for 30 hours, after which plasmids were recovered by alkaline lysis.
(d) Amplify a region containing a spacer sequence and 7 random bases by PCR, attach linkers to both ends of the PCR product, perform next-generation sequencing, and calculate the PAM depletion threshold (PPDV) relative to the empty vector control group, Weblogo was used to generate a PAM sequence that is 5'-NNTA-3' of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium described in this invention.
FIG. 3 is a schematic diagram of protospacer-adjacent-motif-conserved PAM sequences recognized in the prokaryotic system by the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention, and the DNA library obtained by depletion experiments. was analyzed by next-generation sequencing to calculate the PAM depletion threshold (PPDV) relative to the empty vector control group, and the PAM sequence of FrCas9 generated using Weblogo was 5'-NNTA-3'.
Example 3
This example validates the protospacer adjacent motif (PAM) recognized by the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention by interference experiments and demonstrates its prokaryotic-level cleavage ability and A potential gene-editing capability in eukaryotes was confirmed. The results of interference experiments are shown in FIG. A schematic representation of several possible PAM sequences recognized by the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention is shown in FIG.

(1) Materials: pACYC184-FrCas9 obtained in Example 4, target-library plasmid, rudimentary recognized PAM.
(2) Verification method: This example further determined the protospacer adjacent motif (PAM) recognized in the prokaryotic system by the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention by interference experiments. .
Specific operations are as follows.
(a) A total of 16 combinations of NNTA sequences were added to the 3' end of the spacer sequence and cloned into pUC19 from each of the EcoRI and NcoI enzyme cleavage sites to construct a target plasmid.
(b) Each of the 16 types of target plasmids was introduced into E. coli DH5a in the state of competent cells for electroporation containing the FrCas9-related locus, revived at 25 ° C. for 2 hours, and then diluted stepwise, The SOB medium containing both ampicillin sodium (100 μg/ml) and chloramphenicol (34 μg/ml) resistance was applied in dots, incubated overnight at 25° C., and the number of monoclonal bacteria was observed.
FIG. 5C is a schematic representation of an interference experiment of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention. Based on the NNTAs discovered in depletion experiments, target plasmids with different combinations of 16 NNs were constructed, and monoclonal colony numbers were observed by interference experiments. The leftmost column in FIG. 5A is for FrCas9 alone, and the right side is for targeted cleavage of the target plasmid with FrCas9. FIG. 5B is a statistical diagram of the cleavage effects of various PAM sequences recognized by the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention, which shows different combinations of 14 NNs. It showed that the cfu (colony forming unit) of the target plasmid was lower than that of the control group. As is clear from the above results, the protospacer adjacent motif (PAM) recognized by the newly discovered CRISPR/Cas9 system in the prokaryotic system is 5'-NNTA-3' (N is A, T, C, representing any base in G) was verified by interference experiments.
Example 4
In this example, the extent of tracrRNA required to cleave target DNA sequences in the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention was verified by interference experiments. The results of the interference experiment are shown in Figure 5C.

(1) Materials: pACYC184-FrCas9 obtained in Example 5, target plasmid, rudimentary recognized PAM.
(2) Verification method: This example uses an interference experiment to determine the tracrRNA range required for the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention to cleave the target DNA sequence in a prokaryotic system. Confirmed further.
Specific operations are as follows.
(a) A total of 6 gene sequences of all possible noncoding regions (Noncoding) in wild-type Faecalibaculum rodentium are each cloned into target plasmids by Gibson cloning method to construct target-NC 1-6 plasmids. Here, NC6 is the full-length noncoding region concatenated, and NC1-5 represent the first to fifth possible noncoding regions, respectively. Each NC plasmid had a strong heterologous promoter J23119 added upstream of the non-coding region, with "+" representing the forward non-coding region and "-" representing the reverse non-coding region, respectively.

(b) pACYC184-FrCas9 obtained in Example 5 removes all possible non-coding regions by the method of PCR homologous recombination, retains the gene sequences of CRISPR-related proteins and CRISPR arrays, and pACYC184-ΔFrCas9 built.
(c) Each of the 12 target-NC plasmids was introduced into E. coli DH5a in electroporation competent cells containing pACYC184-ΔFrCas9, resuscitated at 25° C. for 2 hours, and then serially diluted, A SOB medium containing both ampicillin sodium (100 μg/ml) and chloramphenicol (34 μg/ml) resistance was applied in dots, incubated overnight at 25° C., and the number of monoclonal bacteria was observed.
Figure 5C is a schematic diagram of the interference experiment of the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention. Clearly less than the case, the first candidate non-coding region was shown to be able to support efficient targeted cleavage of DNA sequences by FrCas9 in E. coli.
Example 5
In this example, ODN experiments verified the ability of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention to cleave target DNA sequences in eukaryotic cells, and the ODN-PCR results were analyzed. FIG. 6A shows the results of Sanger sequencing in FIG. 6B.

(1) Materials: all amino acid sequences, CRISPR array sequences, tracrRNA sequences and recognized PAMs of Faecalibaculum rodentium edited genes obtained in Examples 1-6.
(2) Verification method: In this example, the ability of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention to cleave target DNA sequences exhibited in eukaryotic cells was verified by ODN experiments. .
Specific operations are as follows.
(a) A human-derived optimized FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.
(b) Select 30 bp upstream of NGTA, NATA, NCTA, and NTTA on the human RNF2 gene as the target sequence, introduce it into the CRISPR array by the Gibson method, and add a human-derived eukaryotic strong promoter U6 upstream of the CRISPR array. and constructed the PX330-FrCas9-array plasmid.
(c) A mouse-derived strong eukaryotic promoter U6 was added upstream of the tracrRNA determined in Example 4 to construct a PX330-FrCas9-array-tracrRNA plasmid.
(d) 2.5 μg of the PX330-FrCas9-array-tracrRNA plasmid targeting different gene sites constructed above and 1.5 μl of ODN were introduced into HEK293T cells in good condition by electrotransformation, and 72 After hours, whole cells were harvested and DNA extracted.
(e) ODN-PCR is performed using a pair of primers designed near the RNF2 target gene site and on the ODN sequence, and the presence or absence of bands is observed by agarose gel electrophoresis, and the presence or absence of target cleavage events is rudimentary confirmed. did.
(f) Sanger sequencing verified that the ODN was successfully inserted into the target site, demonstrating that FrCas9 has the ability to edit target DNA in eukaryotic cells.
As a result of ODN-PCR, as shown in FIG. 6A, target bands with correct band sizes were observed in all of NGTA, NATA, NCTA and NTTA. As a result of Sanger sequencing, as shown in FIG. 8, the ODN insertion position is 3 to 4 bp bases upstream of PAM, and FrCas9 and conventional SpCas9 have the same range of occurrence of cleavage of the target gene sequence. I understand.
Example 6
This example verified the optimal length of the sgRNA recognition portion in eukaryotic cells of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention by ODN experiments, and the results are shown in FIG. .

(1) Materials: all amino acid sequences, sgRNA sequences and recognized PAMs of Faecalibaculum rodentium edited genes obtained in Examples 1-6.
(2) Verification method: This example verified the optimal length of the sgRNA recognition portion in eukaryotic cells of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention by ODN experiments. .
Specific operations are as follows.
(a) A human-derived optimized FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.
(B) 30 bp upstream of GGTA near the target site of SpCas9, which was confirmed in previous studies to have a relatively high cleavage effect, was selected as the target sequence, and an sgRNA with a recognition length of 19 to 23 bp was constructed by the Gibson method, and the sgRNA was A human-derived strong eukaryotic promoter U6 was added upstream to construct a PX330-FrCas9-sgRNA plasmid.
(c) 2.5 μg of PX330-FrCas9-sgRNA plasmid targeting different gene sites constructed above and 1.5 ul of ODN were introduced into HEK293T cells in good condition by electrotransformation, and after 72 hours Whole cells were harvested and DNA extracted.
(d) ODN-PCR is performed using a pair of primers designed near the target gene site and on the ODN sequence, and the presence or absence of a band is observed by agarose gel electrophoresis to rudimentarily confirm the presence or absence of the target cleavage event. , compared the intensity of the bands of sgRNAs with recognition lengths of 19-23 bp.
(e) To quantify the Indel rate of target regions, amplicon high-throughput libraries were prepared to compare the cleavage effects of sgRNAs of recognition length 19-23 bp.
As shown in Figure 7, the optimal recognition length of FrCas9 sgRNA was 21bp, 22bp or 23bp.
Example 7
This example validates the optimal length of the sgRNA backbone in eukaryotic cells of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention by ODN experiments and the results are shown in FIG.
(1) Materials: all amino acid sequences, sgRNA sequences and recognized PAMs of Faecalibaculum rodentium edited genes obtained in Examples 1-6.
(2) Validation method: This example validated the optimal length of sgRNA backbone in eukaryotic cells of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention by ODN experiments.
Specific operations are as follows.
(a) A human-derived optimized FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.
(B) 30 bp upstream of GGTA near the target site of SpCas9, which was confirmed in previous studies to have a relatively high cleavage effect, was selected as the target sequence, and an sgRNA with a backbone length of 71 nt to 95 nt was constructed by the Gibson method, and the sgRNA was A human-derived strong eukaryotic promoter U6 was added upstream to construct a PX330-FrCas9-sgRNA plasmid.
(c) 2.5 μg of the PX330-FrCas9-sgRNA plasmid targeting different gene sites constructed above and 1.5 μl of ODN were introduced into HEK293T cells in good condition by electrotransformation, and after 72 hours Whole cells were harvested and DNA extracted.
(d) ODN-PCR is performed using a pair of primers designed near the target gene site and on the ODN sequence, and the presence or absence of a band is observed by agarose gel electrophoresis to rudimentarily confirm the presence or absence of the target cleavage event. , compared the intensity of the bands of sgRNAs with backbone lengths of 71nt to 95nt.
(e) To quantify the Indel rate of target regions, amplicon high-throughput libraries were prepared to compare the cleavage effects of sgRNAs with backbone lengths from 71nt to 95nt.
As shown in Figures 8 and 3, the optimal sgRNA backbone for FrCas9 was between 83nt and 91nt.
Example 8
In this example, by GUIDE-seq experiments, the cutting effect in eukaryotic cells of the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention is higher than SpCas9, and the off-target rate is higher than SpCas9 verified to be low.
(1) Materials: full amino acid sequence of Faecalibaculum rodentium edited gene obtained in Examples 1-6, 22 bp sgRNA sequence, 5′-GGTA-3′ PAM sequence, SpCas9 amino acid sequence, SpCas9 sgRNA sequence and SpCas9 5′- NGG-3' PAM sequence.
(2) Verification method: In this example, according to the GUIDE-seq experiment, the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention has a higher cutting effect in eukaryotic cells than SpCas9, and is turned off. It was verified that the target rate is lower than SpCas9.
Specific operations are as follows.
(a) A human-derived optimized FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.
(B) Select 30 bp upstream of GGTA near the target site of SpCas9, which was confirmed in previous studies to have a relatively high cleavage effect, as a target sequence, construct an sgRNA with a recognition length of 22 bp by the Gibson method, and upstream of sgRNA A human-derived eukaryotic strong promoter U6 was added to construct a PX330-FrCas9-sgRNA plasmid. At the same time, a 20 bp SpCas9 sgRNA PX330-SpCas9-sgRNA plasmid was constructed.
(c) 2.5 μg of PX330-FrCas9-sgRNA plasmid and PX330-SpCas9-sgRNA plasmid targeting different gene sites constructed above, and 1.5 μl of ODN are electrotransformed into HEK293T cells in good condition 72 hours later, all cells were harvested and DNA was extracted.
(d) ODN-PCR is performed using a pair of primers designed near the target gene site and on the ODN sequence, and the presence or absence of a band is observed by agarose gel electrophoresis to rudimentarily confirm the presence or absence of the target cleavage event. , a GUIDE-seq library was prepared for the DNA for which bands were observed.
(e) Bioinformatic analysis compared the on-target cleavage effect and off-target situation of SpCas9 and FrCas9 at the same site.
As shown in FIG. 9, at the DYRK1A-T2 site, the number of on-target reads for FrCas9 was 3257, which was higher than the number of on-target reads for SpCas9 of 2456. At the same time, in FrCas9, no off-target was detected, whereas in SpCas9, three off-target sites were found. In addition, at the GRIB2B-T9 site, the number of on-target reads for FrCas9 was 34,970, which was higher than the number of on-target reads of 20,434 for SpCas9. At the same time, in FrCas9, no off-target was detected, whereas in SpCas9, three off-target sites were found. From the above data, FrCas9 was found to be a Cas9 protein with better cleavage efficiency and specificity than SpCas9.
Example 9 FrCas9 can be used for base editing (BE).
Many human genetic diseases are due to single nucleotide mutations and cannot be treated by traditional methods. Base editors are one of the newest and most effective ways to achieve precise gene editing. characterized by nicking CRISPR-Cas proteins to locate specific target DNA, modifying and mutating such target DNA using DNA deaminase without double-stranded DNA breaks; Make corrections to the lesion bases. Cytosine base editors (CBE) mutate base pairs C:G to T:A in DNA, and adenine base editors (ABE) mutate base pairs A:T to G:C.
First, three point mutations E796A, H1010A, D1013A were introduced into FrCas9 to produce a different FrCas9 nickase (nFrCas9). E796A nFrCas9 was then ligated into the optimized fourth generation cytosine base editor BE4Gam and the seventh generation adenine base editor ABE7.10. It was observed that the editing window of FrCas9-BE4Gam was 6-10 bases (Fig. 10B) and that of FrCas9-ABE7.10 was 6-8 bases (Fig. 10C). Based on the above characteristics, the target ranges of FrCas9-BE4Gam and FrCas9-ABE7.10 in the ClinVar database were calculated. For pathogenic mutations that could be correctly corrected by FrCas9-BE4Gam, 90.38% (235/260) editing events differed from SpCas9-BE4Gam. For pathogenic mutations that can be correctly corrected by FrCas9-ABE7.10, 92.21% (1196/1297) editing events differed from SpCas9-ABE7.10 (Fig. 10D). Thus, FrCas9 has greatly expanded its targeting in the human genome by TA-rich PAMs and has been used as a base editor to correct mutations associated with human disease.

Also, the PAM of FrCas9(5'-NNTA-3') is palindromic, which allows the sgRNAs to exist in pairs "back to back" (Fig. 10E). This feature expands the range of the FrCas9 base editor by modifying two adjacent editing windows simultaneously (Fig. 10E) and can increase target distribution and density of FrCas9 sgRNAs. The distribution of 5'-GG-3' (representing SpCas9 PAM) and 5'-TA-3' (representing FrCas9 PAM) in the human genome was calculated (Figures 11A and 11B). Compared to SpCas9 (median=5 bp, mean=8.66 bp)5, FrCas9 exhibits a more dense distribution in the human genome (median=1 bp, mean=6.16 bp), providing additional sites of application did.
Example 10 FrCas9 targets the TATA box to regulate gene expression and can be an effective tool for CRISPR activation and repression.
A TATA box (TATAbox/Hognessbox) is one of elements that constitute a eukaryotic promoter. The matching order is TATA(A/T) A(A/T) (non-template strand sequence). It is located approximately −30 bp (−25 to −32 bp) upstream of the transcription initiation site of many eukaryotic genes and consists essentially of the base pair AT and is the selection that determines the transcription initiation of genes. , is one of the binding sites for RNA polymerase, and transcription is initiated after the RNA polymerase and the TATA box bind tightly. Since the PAM sequence of FrCas9 is a NNTA, it has a natural advantage in targeting the TATA box.
We targeted the TATA box in three genes, ABCA1, UCP3 and RANKL, to test the ability of FrCas9 CRISPR interference (CRISPRi) (Fig. 12A). It involved direct utilization of the active form of FrCas9 to target and cleave the TATA box to disrupt the TATA box. As a result, FrCas9 reduced the expression of ABCA1, UCP3 and RANKL by 31.37%, 49.91% and 39.62%, respectively. In addition, dFrCas9 without cleavage activity was used to bind directly to the TATA box. As a result, the expression of ABCA1, UCP3 and RANKL decreased by 61.67%, 45.61% and 42.60% respectively (Fig. 12B). As such, FrCas9 was found to have unique potential in precisely regulating gene expression.
We also tested the effect of FrCas9 CRISPR activation (CRISPRa) using dFrCas9-VP64, which targets the TATA box directly, and compared its performance to dSpCas9-VP64, which targets the TATA box upstream. These experiments were performed with the ABCA1, SOD1, GH1, BLM2 genes. As a result, it was found that dFrCas9-VP64 can achieve effective transcriptional activation. Also, in ABCA1, GH1, BLM2, the activation fold of dFrCas9-VP64 is higher than dSpCas9-VP64, and in the SOD1 gene, the activation fold of dFrCas9-VP64 is equivalent to dSpCas9-VP64 (Fig. 12C). Therefore, FrCas9 proved to be a promising CRISPR screening tool due to its unique 5'-NNTA-3'PAM.
Example 11 FrCas9 can be used for precision gene editing (Prime Editing, PE).
Prime Editing (PE) is a completely new precision gene editing tool. This technology can achieve free substitution of single nucleotides and accurate insertion/deletion of multiple nucleotides, greatly reducing harmful by-products generated by indels during gene editing, significantly improving editing accuracy, It is considered to be a major advance in gene editing that has the potential to overcome fundamental obstacles to treating genetic diseases with existing gene-editing methods. The PE system contains two parts, a modified guide RNA (pegRNA) and a prime editor protein. The pegRNA has a dual function, being able to direct the editing protein to the target site as well as containing the template sequence for editing. The prime editor protein is a fusion of a mutated Cas9 nickase (which can cut only one strand of DNA) and reverse transcriptase. After cleaving the target site with Cas9, the reverse transcriptase reverse transcribes the pegRNA as a template, thereby incorporating the necessary edits into the DNA strand, the modified sequences preferentially displacing the original genomic DNA, and the target A permanent edit was made to the DNA at the position (Fig. 13).
Based on the biological properties of FrCas9, fuse FrCas9 with reverse transcriptase, design corresponding pegRNA based on its PAM sequence, construct FrCas9-PE gene editing system, and optimize its gene editing performance did. The pegRNA designed based on the FrCas9 PAM sequence had two sequences added to the 3' end sequence of the pegRNA compared to the sgRNA. The first sequence is a primer binding site (PBS), capable of complementary binding to the ends of the cleaved target DNA strand, thereby initiating the reverse transcription process. The second sequence is a reverse transcription template (RT template) with targeted point mutations or insertion-deletion mutations to achieve precise gene editing. Previous studies have shown that the sequence lengths of PBS and RT templates significantly affected the gene editing efficiency of the PE system and differed by gene site. Therefore, optimization of the editing efficiency was investigated by combining different lengths of PBS and RT template sequences.
At the cellular level, the gene-editing effects of the FrCas9-PE system were rudimentary verified. In HEK293T cells, we verified the gene-editing function of the FrCas9-PE system by targeting the HEK293T-RNF2 gene site, which is often used to evaluate the gene-editing efficiency of CRISPR. Current experimental results indicated that FrCas9-PE can exert a base-editing effect at specific sites. After that, the existing FrCas9-PE was further modified such as codon optimization, optimization of the position and number of nuclear localization sequences, and reverse transcriptase modification. Finally, a novel FrCas9-PE gene-editing system with high base-editing efficiency, low off-target reactions, and low gene-editing by-products (indel) was developed (FIG. 14).

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

Gene editing technologies allow for the remodeling of DNA sequence locations, such as the first generation of gene editing tools zinc finger nucleases (ZFNs) and the second generation of gene editing tools, zinc finger nucleases (ZFNs). Gene-editing tools, transcription activator-like effector nucleases (TALENs), can all be used to modify target genomes, but these methods are difficult to design, difficult to produce, and costly. And there is a problem that versatility is not good.
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR-associated protein) system is an innate immune system derived from archaea and bacteria. It is a system and a third generation gene editing tool. Unlike conventional gene-editing tools (protein-DNA recognition), this method utilizes the principle of nucleobase pair complementarity to recognize target DNA sequences and Cas effector proteins are site-specific. It is highly adaptable, simple in design, low in cost and high in efficiency. Cas proteins contain a variety of different effector domains that play roles in a variety of activities such as nucleic acid recognition, structural stabilization of complexes, and hydrolysis of phosphodiester bonds in DNA. Among them, the type II CRISPR/Cas9 system (Streptococcus pyogene Cas, SpCas9) derived from Streptococcus pyogenes is currently the most widely used CRISPR/Cas system due to its high cleavage efficiency. This system recognizes and cleaves the protospacer adjacent motif (PAM) sequence "NGG" in the target polynucleotide, leaving a single blunt or sticky end, affecting gene editing.
The vast and diverse metagenome harbors uncultivated or undiscovered microbes, the potential for many undiscovered CRISPR/Cas9 systems, and the prokaryotic and eukaryotic evolution of these systems. It is necessary to confirm the activity in organisms and in vitro environments.
In 2015, Dr. Byoung-Chan Kim's team isolated a new anaerobic strain ALO17 from the feces of laboratory mice C57BL/6J, and by phylogenetic analysis of the sequence of the prokaryotic 16S rRNA gene, the strain was named Holdemanellabiformis DSM 3989T. , Faecalicoccus pleomorphus ATCC 29734T, Faecalitaleacylindroides ATCC 27803T, and Allobaculumstercoricanis DSM 13633T (sequence identities of 87.4%, 87.3% and 8%, respectively). 6.9%, and 86 .9%). Based on a large body of taxonomic evidence, this species has been assigned a new genus in the family Erysipelothricaceae, and Faecalibaculum rodentium gen. nov. , sp. nov. was named. Over the next five years, scientists around the world have studied this strain in two areas: gut microbiome and high-fat diet, gut microbiome and tumorigenesis, but nothing has yet been reported on gene editing. do not have.

The present invention overcomes the deficiencies of the prior art, has new physicochemical properties, and is capable of producing multiple different PAM (protospacer adjacent motifs) including NGTA and NNTA (N represents A, C, G or T). It is an object of the present invention to provide a type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium that is capable of recognizing Flanking Motif) sequences.
In order to achieve the above objects, the technical means adopted in the present invention are:
A type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium, comprising Cas9 protein, accessory protein, crRNA (i.e. CRISPR RNA) and tracrRNA (i.e. transactivating CRISPR RNA), wherein Cas9 functions in the form of an RNP complex (i.e., a ribonucleoprotein complex) of a protein and a guide RNA formed by binding tracrRNA to crRNA,
The Cas9 protein is a DNA endonuclease, and the Cas9 protein is the amino acid sequence shown in SEQ ID NO: 1, or at least 80%, 85%, 90%, 95%, 98% of the amino acid sequence shown in SEQ ID NO: 1 %, or 99% homology.
Said Cas9 protein cleaves double-stranded DNA complementary to crRNA upstream of the PAM sequence with a nuclease domain, said nuclease domain being an HNH-like nuclease domain and/or a RuvC-like nuclease domain.
In some embodiments, the "ribonucleoprotein complex" is preferably the "FrCas9 protein complex" in the present invention.
The mutational status of some key amino acid positions of specific Cas9 proteins is described. Mutation of E at amino acid position 796 to A results in a Nickase nuclease, mutation of N at amino acid position 902 to A results in a Nickase nuclease, H at amino acid position 1010 to A. Mutation results in a nickase nuclease, mutation of D at amino acid position 1013 to A results in nickase nuclease, and mutation of E at amino acid position 796 and D at amino acid position 1013 to A at the same time. It results in a Cas9 nuclease that is incapable of cleaving but retains binding ability (ie, inactive Cas9 (dead Cas9)).
The accessory proteins include Cas1 accessory protein, Cas2 accessory protein and Csn2 accessory protein,
The Cas1 accessory protein is the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence set forth in SEQ ID NO: 2 has an array,
The Cas2 accessory protein is the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence set forth in SEQ ID NO: 3 has an array,
The Csn2 accessory protein is the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence set forth in SEQ ID NO: 4 has an array.
The CRISPR RNA is generated by transcription from a CRISPR array, and the CRISPR RNA is at least 80%, 85%, 90%, 95% of the RNA sequence set forth in SEQ ID NO:5 or the nucleic acid sequence set forth in SEQ ID NO:5. , 98% or 99% homology, wherein the CRISPR array comprises a direct repeat sequence and a spacer sequence, wherein the direct repeat sequence is shown in SEQ ID NO: 6 a nucleic acid sequence or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the nucleic acid sequence set forth in SEQ ID NO: 6, wherein the spacer sequence comprises 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 shown in SEQ ID NO:7.
The transactivating CRISPR RNA contains a sequence complementary to a direct repeat sequence of CRISPR RNA, and the transactivating CRISPR RNA is the nucleic acid sequence shown in SEQ ID NO: 8, or the nucleic acid shown in SEQ ID NO: 8 Have a nucleic acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homologous to the sequence.
The backbone of the guide RNA, which is crRNA bound to tracrRNA, is composed of 7-24 nt of the crRNA direct repeat sequence and the corresponding length of the tracrRNA sequence, and these two parts are designated as "GAAA", "TGAA", can be fused via a linker such as "AAAC" to form an sgRNA backbone, preferably a sgRNA backbone formed by fusing a 20 nt direct repeat sequence and a 71 nt tracrRNA sequence via a "GAAA" is shown in SEQ ID NO: 9, and further preferred variants based on the sgRNA backbone are:
an sgRNA backbone formed by fusing the first 18-14 nt of the direct repeat sequence of the crRNA and the last 69-65 nt of the tracrRNA through a linker sequence (such as "GAAA"), which further comprises
(a) a backbone represented by SEQ ID NO: 10, which has a length of 91 nt and contains a direct repeat sequence of 18 nt and a tracrRNA of 69 nt;
(b) an sgRNA backbone having a length of 89 nt, containing a direct repeat sequence of 17 nt and a tracrRNA of 68 nt, and represented by SEQ ID NO: 11;
(c) an sgRNA backbone of 87 nt in length, containing a 16 nt direct repeat sequence and 67 nt of tracrRNA, and represented by SEQ ID NO: 12;
(d) an sgRNA backbone having a length of 85 nt, containing a direct repeat sequence of 15 nt and a tracrRNA of 66 nt, and represented by SEQ ID NO: 13;
(e) a backbone represented by SEQ ID NO: 14, which has a length of 83 nt and contains a direct repeat sequence of 14 nt and a tracrRNA of 65 nt;
A skeleton selected from the group consisting of
The sgRNA backbone may have a nucleic acid sequence with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% homology to any one of SEQ ID NOs:9-14.
The type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium binds or cleaves specific DNA in a biological process (e.g., gene editing). The range of the length of the binding portion that is realized by recognizing the guide RNA by complementing and pairing with the target site of the specific DNA, and the pairing portion of the guide RNA and the target site of the specific DNA is , 14-30 bp (preferably 20-23 bp), and the specific DNA is prokaryotic or eukaryotic DNA.
The length of the paired binding portion of the guide RNA and the specific DNA target site is 21 bp, 22 bp or 23 bp, wherein the RNP protein complex is adjacent to a protospacer adjacent motif (PAM). It is very sensitive to 14 bp base mismatches, said 14 bp being the seed region.
The protospacer-adjacent motif sequence required for the DNA binding or cleaving function is 5'-NNTA-3' downstream of the guide RNA/sgRNA recognition sequence.
The present invention further provides a CRISPR/Cas9 gene editing system for editing or joining DNA, activating CRISPR or interfering with CRISPR, wherein the CRISPR/Cas9 gene editing system comprises the type II CRISPR/Cas9 gene Editing system. Preferably, said DNA is prokaryotic or eukaryotic DNA. Preferably, said type II CRISPR/Cas9 gene editing system is derived from Faecalibaculum rodentium.
In another aspect, the present invention also provides a CRISPR/Cas9 gene editing system for the production of Nickase, Inactive Cas9, Base Editor or Prime Editor, wherein said CRISPR/Cas9 The gene editing system is the Type II CRISPR/Cas9 gene editing system described above. Preferably, the process of producing the nickase, inactive Cas9, Base Editor or Prime Editor is performed in a prokaryote or eukaryote.
The beneficial effects of the present invention compared with the prior art are as follows.
(1) The present invention discovers a type II CRSIPR/Cas9 gene editing system in Faecalibaculum rodentium through bioinformatics analysis, and the gene editing system can be applied to edit prokaryotic or eukaryotic genes, gene editing tools Offer new options in the box.
(2) The type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium provided by the present invention has new physicochemical properties, can recognize a plurality of different PAM sequences, and the gene editing system A specific sequence of PAM that is recognized is 5'-NNTA-3' downstream of the guide RNA/sgRNA recognition sequence.
(3) The type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium provided by the present invention has a higher cutting efficiency at the same DNA site than the most commonly used SpCas9, and the most commonly used off-target rate It is a safer and more effective gene-editing tool than SpCas9.
(4) In the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium provided by the present invention, the PAM recognized by the gene editing system has palindrome characteristics, and the target site is on the genome distributed "back-to-back" in , with a higher density than SpCas9.
(5) The type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium provided by the present invention has high flexibility and can be remodeled into a base editor (Base Editor) and a prime editor (Prime Editor), It is a gene editing tool that can be applied in different scenarios.

1 is a schematic diagram of the type II CRISPR/Cas9 gene editing system of Faecalibaculum rodentium according to the present invention. FIG. Fig. 2 is a structural schematic diagram of the Cas9 protein of the type II CRISPR/Cas9 gene editing system of Faecalibaculum rodentium according to the present invention; FIG. 2 is a schematic diagram of an optimal sgRNA backbone and a predicted RNA secondary structure of a guide RNA molecule recognized by the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 3A shows the full length of the 95nt gRNA. FIG. 3B shows a gRNA with a backbone length of 91 nt, 18 nt direct repeat plus 69 nt tracrRNA. FIG. 3C shows an sgRNA with a backbone length of 89 nt, 17 nt direct repeat plus 68 nt tracrRNA. FIG. 3D shows a gRNA with a backbone length of 87 nt, 16 nt direct repeat plus 67 nt tracrRNA. FIG. 3E shows an sgRNA with a backbone length of 85 nt, 15 nt direct repeat plus 66 nt tracrRNA. FIG. 3F shows an sgRNA with a backbone length of 83 nt, 14 nt direct repeat plus 65 nt tracrRNA. FIG. 2 is a schematic representation of the prokaryotic PAM sequence of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the invention. FIG. 2 is a schematic diagram of a prokaryotic interference experiment of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 2 is a schematic representation of eukaryotic cleavage of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 6A is an ODN-PCR gel electropherogram. FIG. 6B is a peak diagram from Sanger sequencing. FIG. 2 is a schematic diagram of the optimal length of sgRNA for the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 7A shows cleavage efficiency of different sgRNA lengths at the HEK293 SITE2-T2 site. Figure 7B shows the cleavage efficiency of different sgRNA lengths at the DNMT1-T3 site. FIG. 7C shows the cleavage efficiency of different sgRNA lengths at the RNF2-T6 site. Fig. 2 shows the optimal length of the recognition sequence of sgRNA of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention. FIG. 3 is a schematic diagram showing a comparison of the target efficiency and off-target in GUIDE-seq of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention and the target efficiency and off-target in SpCas9 in GUIDE-seq. FrCas9-BE base editor experimental results. Here, FIG. 10A is a principle diagram and results for confirming the form of FrCas9 nickase by ODN breakpoint PCR. FIG. 10B is the edit window of nFrCas9-BE4Gram. FIG. 10C is the edit window of nFrCas9-ABE7.10. FIG. 10D is a Venn diagram of the unique pathogenic mutations that can be corrected with the SpCas9 and FrCas9 base editors in the ClinVar database. FIG. 10E shows C>T editing efficiency of FrCas9-BE4Gam using two “back-to-back” sgRNAs simultaneously. FIG. 2 is a distribution map of target sites of FrCas9 and SpCas9 in the human genome. Here, Figure 11A shows the distribution of 5'-GG-3' (representing SpCas9 PAM) in the GRCh38 human genome. Figure 11B shows the distribution of 5'-TA-3' (representing FrCas9 PAM) in the GRCh38 human genome. FIG. 4 shows the application of FrCas9 specifically targeting the TATA box to CRISPR interference and CRISPR activation. Here, FIG. 12A is a schematic diagram of the TATA box of the ABCA1 gene, which is the target of FrCas9. FIG. 12B shows CRISPR interference in FrCas9 and SpCas9 by targeting the TATA box and CRISPR activation in FrCas9 and SpCas9 by targeting the TATA box. FIG. 12C shows CRISPR activation in FrCas9 and SpCas9 by targeting the TATA box. Schematic diagram of a gene editing system for prime editing. FIG. 4 shows validation of the gene-editing action of the FrCas9-PE prime editing system by Sanger sequencing. The measured sequence is CGAACACCTCAGGTAAT (SEQ ID NO:33).

In order to better describe the purpose, technical solution and advantages of the present invention, the present invention is further described below based on specific examples.
The type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium contains Cas9 protein, accessory proteins, CRISPR RNA and transactivating CRISPR RNA, and as shown in FIG. 1, the Cas9 protein is a DNA endonuclease. wherein the Cas9 protein has at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 1 It has an amino acid sequence.
As shown in Figure 2, as a preferred embodiment of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention, the Cas9 protein is complemented with crRNA upstream of the PAM sequence by various nuclease domains. The various nuclease domains are HNH-like nuclease domains or RuvC-like nuclease domains.
The Cas9 protein (Faecalibaculum rodentium Cas9) according to the present invention, sometimes simply called FrCas9 protein, contains 1372 amino acids (SEQ ID NO: 1) and is a multidomain and multifunctional DNA endonuclease. The Cas9 protein efficiently cleaves double-stranded DNA complementary to sgRNA upstream of the PAM by means of various nuclease domains. For example, the HNH-like nuclease domain cleaves the DNA strand complementary to the sgRNA sequence, and the RuvC-like nuclease domain cleaves the non-complementary DNA strand. Here, mutation of E at amino acid position 796 to A results in a nickase nuclease, mutation of N at amino acid position 902 to A results in a nickase nuclease, and H at amino acid position 1010 to A. Mutation of D at amino acid position 1013 to A results in nickase nuclease, E at amino acid position 796 and D at amino acid position 1013 are simultaneously mutated to A This results in a Cas9 nuclease that is incapable of cutting but retains binding ability.
As a preferred embodiment of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention, said accessory proteins comprise a Cas1 accessory protein, a Cas2 accessory protein and a Csn2 accessory protein, wherein said Cas1 accessory protein has the sequence Having an amino acid sequence represented by number 2 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence represented by SEQ ID NO: 2, said Cas2 The accessory protein has the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:3. wherein the Csn2 accessory protein has the amino acid sequence set forth in SEQ ID NO:4 or at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:4 has an amino acid sequence with
The accessory proteins Cas1, Cas2, Csn2 according to the invention are involved in foreign gene capture and maturation of crRNA.
As a preferred embodiment of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention, the CRISPR RNA is produced by being transcribed from a CRISPR array, and the CRISPR RNA is the RNA shown in SEQ ID NO: 5 wherein said CRISPR array comprises direct repeat sequences and spacer sequences, said direct repeat sequences being shown in SEQ ID NO:6 and said spacer sequences being shown in SEQ ID NO:7.
The CRISPR RNAs (crRNAs) described in the present invention exploit base-pair complementarity to induce Cas proteins to recognize invading foreign genomes. Bacteria, when exposed to bacteriophages, viruses, etc., provide a genetic record of infection by incorporating short pieces of foreign DNA as new spacers between CRISPR repeat spacer sequences in the host chromosome; When a foreign gene re-invades the organism, a crRNA precursor (pre-crRNA) is generated by being transcribed from the CRISPR array, and its 5′ end is complementary to the sequence of the gene that has invaded from outside, and is 30 bp in length. A spacer sequence has a repeat sequence 36 bp in length at the 3' end.
As a preferred embodiment of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention, said transactivating CRISPR RNA contains a sequence complementary to a CRISPR RNA direct repeat sequence, and said transactivating A type CRISPR RNA is shown in SEQ ID NO:8.
Trans-activating crRNAs (tracrRNAs) according to the present invention are RNAs that do not encode proteins and are involved in maturation of crRNAs and formation of sgRNAs. The pre-crRNA cleaves 0-16 nt upstream of the spacer sequence and 12-29 nt downstream of the repeat sequence by the action of tracrRNA and Cas9 nuclease to form mature crRNA, which further binds to tracrRNA to form a tracrRNA-crRNA complex. form the body. The complex is 14-30 bp in length and contains a portion that recognizes a sequence of foreign DNA. By adding a tetraloop of four bases, such as a "GAAA", "TGAA" or "AAAC" sequence, between the crRNA downstream and the tracrRNA upstream, the tracrRNA and crRNA are linked to form an sgRNA. can be formed. Such sgRNAs contain two bulge structures and three duplex structures upstream, and one stem loop structure downstream, and also recognize sequences of foreign DNA. It can be divided into partial and skeletal parts.
By adjusting the length of the portion of the sgRNA that recognizes the foreign DNA sequence and the length of the tracrRNA, the cleavage action of the endonuclease can be further optimized. From previous experiments of the present invention, the optimal length of the portion of the sgRNA that recognizes the foreign DNA sequence is 21 bp, 22 bp or 23 bp (FIG. 8), and the optimal length of the backbone portion is shown in FIGS. 7, 91 nt (18 nt crRNA direct repeat + 69 nt tracrRNA), 89 nt (17 nt crRNA direct repeat + 68 nt tracrRNA), 87 nt (16 nt crRNA direct repeat + 67 nt tracrRNA), 85 nt (15 nt crRNA It was proved that there are five types of 83 nt (direct repeat sequence + 66 nt tracrRNA) and 83 nt (14 nt crRNA direct repeat sequence + 65 nt tracrRNA).
In a preferred embodiment of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention, said type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium binds DNA in a biological process or disconnect.
In a preferred embodiment of the Faecalibaculum rodentium type II CRISPR/Cas9 gene editing system according to the present invention, said DNA is prokaryotic or eukaryotic DNA.
In some embodiments, the FrCas9 of the present invention can recognize multiple types of protospacer adjacent motifs (PAMs) flanking the downstream side of the target sequence to cause DNA double-stranded molecular breaks (DSBs). can. Recognition of the target sequence by the FrCas9 protein requires two key elements: the nucleotides complementary to the crRNA spacer sequence and the Protospacer Adjacent Motif (PAM) flanking the complementary sequence. As shown in Figure 4, depletion experiments revealed that FrCas9 has a cleaving effect in the prokaryotic system, with positions 3 and 4 of the PAM sequence recognized by this newly discovered type II CRISPR/Cas9 system It was rudimentary verified to be TA. Furthermore, as shown in Figure 5, interference experiments demonstrated that the PAM downstream of the target sequence recognized by FrCas9 was 5'-NNTA-3'. As shown in FIG. 6, all of the above PAMs were verified in eukaryotic experiments. By artificially designing spacer sequences in crRNA, such CRISPR-Cas9 systems can target almost any DNA sequence of interest in the genome, generating site-specific blunt ends. produces a double-strand break (DSB). By repairing the DSB with non-homologous end joining, small random insertions/deletions (indels) at the break site are generated to inactivate the gene of interest. Alternatively, high-fidelity homologous recombination repair can be used to make precise genomic modifications at DSB sites using homologous recombination repair templates.
Many human genetic diseases are due to single nucleotide mutations and cannot be cured by traditional methods. Base editors are one of the newest and most effective ways to achieve precise gene editing. It features a nicking version of the CRISPR-Cas protein that locates specific target DNA and uses DNA deaminase to modify and modify such target DNA without causing double-stranded DNA breaks. Mutate and correct for the diseased base. In some embodiments, the E796A nFrCas9-BEGam cytosine base editor combines E796A nFrCas9 with the optimized fourth generation cytidine base editor BE4Gam to mutate base pairs C:G to T:A in DNA ( E796A nFrCas9-BABE7.10 adenine base editor (ABE) that combines E796A nFrCas9 with the seventh generation adenine base editor ABE7.10 to mutate the base pair A:T to G:C. ) can be constructed.
In some embodiments, the specific PAM of FrCas9 of the present invention is NNTA and has a target site specific to the TATA box (one of the elements that make up the eukaryotic promoter), so FrCas9 is specific By specifically targeting the TATA box, unique CRISPR interference (CRISPRi)/CRISPR activation (CRISPRa) effects can be exerted. Here, CRISPRi either utilizes active FrCas9 to directly target cleave the TATA box to destroy it, or dFrCas9 without cleavage activity (i.e., inactive Cas9) directly binds to the TATA box. It can be realized by letting CRISPRa can be achieved by, but not limited to, dFrCas9-VP64 targeting the TATA box site.
Prime Editing (PE) is an entirely new precision gene editing tool. This technology enables free substitution of single bases and precise insertions and deletions of multiple bases, greatly reducing harmful by-products produced by indels in the process of gene editing, and improving editing accuracy. can be greatly improved and is widely considered to be a significant advance in gene editing. In some embodiments, the FrCas9 of the present invention can be fused with reverse transcriptase and the corresponding pegRNA can be designed based on its PAM sequence to construct the FrCas9-PE gene editing system. Specifically, two sequences are added to the 3' end sequence of pegRNA. The first sequence is the primer binding site (PBS) and can complement the ends of the cleaved target DNA strand to initiate the reverse transcription process, and the second sequence is the reverse transcription template (RT templates) and have targeted point mutations or indel mutations to achieve precise gene editing.
It is a further object of the present invention to provide the use of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium in prokaryotic or eukaryotic gene editing, CRISPR activation or CRISPR interference.
As a preferred embodiment of the use according to the invention, the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium is used to join or cleave DNA at the DNA level.
Example 1
In this example, the RNA secondary structure of the guide RNA molecule recognized by the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention is predicted, and the RNA transcribed from the tracrRNA and the repetitive sequence is predicted. By simulating the binding process, the RNA structure after binding of both was predicted, and the obtained RNA secondary structure is shown in FIG.
(1) Materials: predicted tracrRNA and repeat sequences, and predicted anti-repeat sequences.
(2) Software: NUPACK (http://www.nupack.org/partition/new)
(3) Prediction method: NUPACK, an online application, simulates the in vitro interaction process of 1 µl of each of the two RNAs at 37°C, and predicts the secondary structure of the resulting RNA composition. An RNA secondary structure as shown in 2 was obtained.
As can be seen from FIG. 3, the pre-crRNA is excised by tracrRNA and Cas9 nuclease from 0-16 nt upstream of the spacer sequence and 12-29 nt downstream of the repeat sequence to form the mature crRNA and fuse with the tracrRNA. A tracrRNA-crRNA complex was formed. This complex contained a sequence of foreign DNA complementary to the spacer sequence, with a sequence length of 14-30 bp. Adding a 4-nt tetraloop between the crRNA downstream and the tracrRNA upstream connects them to form two bulge and three double helix structures upstream and three stem-loop structures downstream. can form an sgRNA comprising
Example 2
In this example, the protospacer adjacent motif (PAM) recognized in the prokaryotic system by the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention was found to be 5'-NNTA-3'. .
(1) Materials: genes related to the CRISPR/Cas9 gene editing system predicted by the above examples.
(2) Verification method: In this example, a prokaryotic verification system was constructed for the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention, the cleavage effect was verified, and the recognized PAM sequence was preliminarily searched by second generation sequencing technology and the results are shown in FIG.
The detailed flow is as follows.
(a) Type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention, comprising endonuclease Cas9, accessory proteins Cas1, Cas2, Csn2, CRISPR array and non-coding RNA tracrRNA ) was inserted into the pACYC184 vector, E. coli codon-optimized for the Cas9 protein, the CRISPR array was added with the native spacer sequence and the spacer sequence in the library, and the Cas9 protein, a strong heterologous promoter upstream of the CRISPR array. (heterologous promoter) J23119 was added to construct a prokaryotic expression pACYC184-FrCas9 plasmid.
(b) adding 7 random bases (total of 16384 inserts) to the 3′ end of the spacer sequence of the library and cleaving the multiple cloning site (MCS) of the pUC19 vector with two enzymes, EcoRI and NcoI sites; Sites were selected and the library cloned into a vector to construct the target-library plasmid.
(c) Both the pACYC184 plasmid containing pACYC184-FrCas9 or the empty vector and the target-library plasmid were introduced into E. coli DH5a by the electrotransformation method, revived at 25°C for 2 hours, and then treated with ampicillin sodium (100 µg/ml). and chloramphenicol (34 μg/ml), and incubated at 25° C. for 30 hours, after which plasmids were recovered by alkaline lysis.
(d) Amplify a region containing a spacer sequence and 7 random bases by PCR, attach linkers to both ends of the PCR product, perform next-generation sequencing, and calculate the PAM depletion threshold (PPDV) relative to the empty vector control group, Weblogo was used to generate a PAM sequence that is 5'-NNTA-3' of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium described in this invention.
FIG. 3 is a schematic diagram of protospacer-adjacent-motif-conserved PAM sequences recognized in the prokaryotic system by the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention, and the DNA library obtained by depletion experiments. was analyzed by next-generation sequencing to calculate the PAM depletion threshold (PPDV) relative to the empty vector control group, and the PAM sequence of FrCas9 generated using Weblogo was 5'-NNTA-3'.
Example 3
This example validates the protospacer adjacent motif (PAM) recognized by the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention by interference experiments and demonstrates its prokaryotic-level cleavage ability and A potential gene-editing capability in eukaryotes was confirmed. The results of interference experiments are shown in FIG. A schematic representation of several possible PAM sequences recognized by the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention is shown in FIG.

(1) Materials: pACYC184-FrCas9 obtained in Example 2 , target-library plasmid, rudimentary recognized PAM.
(2) Verification method: This example further determined the protospacer adjacent motif (PAM) recognized in the prokaryotic system by the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention by interference experiments. .
Specific operations are as follows.
(a) A total of 16 combinations of NNTA sequences were added to the 3' end of the spacer sequence and cloned into pUC19 from each of the EcoRI and NcoI enzyme cleavage sites to construct a target plasmid.
(b) Each of the 16 types of target plasmids was introduced into E. coli DH5a in the state of competent cells for electroporation containing the FrCas9-related locus, revived at 25 ° C. for 2 hours, and then diluted stepwise, The SOB medium containing both ampicillin sodium (100 μg/ml) and chloramphenicol (34 μg/ml) resistance was applied in dots, incubated overnight at 25° C., and the number of monoclonal bacteria was observed.
FIG. 5C is a schematic representation of an interference experiment of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention. Based on the NNTAs discovered in depletion experiments, target plasmids with different combinations of 16 NNs were constructed, and monoclonal colony numbers were observed by interference experiments. The leftmost column in FIG. 5A is for FrCas9 alone, and the right side is for targeted cleavage of the target plasmid with FrCas9. FIG. 5B is a statistical diagram of the cleavage effects of various PAM sequences recognized by the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention, which shows different combinations of 14 NNs. It showed that the cfu (colony forming unit) of the target plasmid was lower than that of the control group. As is clear from the above results, the protospacer adjacent motif (PAM) recognized by the newly discovered CRISPR/Cas9 system in the prokaryotic system is 5'-NNTA-3' (N is A, T, C, representing any base in G) was verified by interference experiments.
Example 4
In this example, the extent of tracrRNA required to cleave target DNA sequences in the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention was verified by interference experiments. The results of the interference experiment are shown in Figure 5C.

(1) Materials: pACYC184-FrCas9 obtained in Example 2 , target plasmid, rudimentary recognized PAM.
(2) Verification method: This example uses an interference experiment to determine the tracrRNA range necessary for the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention to cleave the target DNA sequence in a prokaryotic system. Confirmed further.
Specific operations are as follows.
(a) A total of 6 gene sequences of all possible noncoding regions (Noncoding) in wild-type Faecalibaculum rodentium are each cloned into target plasmids by Gibson cloning method to construct target-NC 1-6 plasmids. Here, NC6 is the full-length noncoding region concatenated, and NC1-5 represent the first to fifth possible noncoding regions, respectively. Each NC plasmid had a strong heterologous promoter J23119 added upstream of the non-coding region, with "+" representing the forward non-coding region and "-" representing the reverse non-coding region.

(b) pACYC184-FrCas9 obtained in Example 2 removes all possible non-coding regions by the method of PCR homologous recombination, retains the gene sequences of CRISPR-related proteins and CRISPR arrays, and pACYC184-ΔFrCas9 built.
(c) Each of the 12 types of target-NC plasmids was introduced into E. coli DH5a in electroporation competent cells containing pACYC184-ΔFrCas9, resuscitated at 25° C. for 2 hours, and then serially diluted, A SOB medium containing both ampicillin sodium (100 μg/ml) and chloramphenicol (34 μg/ml) resistance was applied in dots, incubated overnight at 25° C., and the number of monoclonal bacteria was observed.
Figure 5C is a schematic diagram of the interference experiment of the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention. Clearly less than the case, the first candidate non-coding region was shown to be able to support efficient targeted cleavage of DNA sequences by FrCas9 in E. coli.
Example 5
In this example, ODN experiments verified the ability of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention to cleave target DNA sequences in eukaryotic cells, and ODN-PCR results were analyzed. FIG. 6A shows the results of Sanger sequencing in FIG. 6B.

(1) Materials: all amino acid sequences, CRISPR array sequences, tracrRNA sequences and recognized PAMs of Faecalibaculum rodentium edited genes obtained in Examples 1-4 .
(2) Verification method: In this example, the ability of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention to cleave target DNA sequences exhibited in eukaryotic cells was verified by ODN experiments. .
Specific operations are as follows.
(a) A human-derived optimized FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.
(b) Select 30 bp upstream of NGTA, NATA, NCTA, and NTTA on the human RNF2 gene as the target sequence, introduce it into the CRISPR array by the Gibson method, and add a human-derived eukaryotic strong promoter U6 upstream of the CRISPR array. and constructed the PX330-FrCas9-array plasmid.
(c) A mouse-derived strong eukaryotic promoter U6 was added upstream of the tracrRNA determined in Example 4 to construct a PX330-FrCas9-array-tracrRNA plasmid.
(d) 2.5 μg of the PX330-FrCas9-array-tracrRNA plasmid targeting different gene sites constructed above and 1.5 μl of ODN were introduced into HEK293T cells in good condition by electrotransformation, and 72 After hours, whole cells were harvested and DNA extracted.
(e) ODN-PCR is performed using a pair of primers designed near the RNF2 target gene site and on the ODN sequence, and the presence or absence of bands is observed by agarose gel electrophoresis, and the presence or absence of target cleavage events is rudimentary confirmed. did.
(f) Sanger sequencing verified that the ODN was successfully inserted into the target site, demonstrating that FrCas9 has the ability to edit target DNA in eukaryotic cells.
As a result of ODN-PCR, as shown in FIG. 6A, target bands with correct band sizes were observed in all of NGTA, NATA, NCTA and NTTA. As a result of Sanger sequencing, as shown in FIG. 8, the ODN insertion position is 3 to 4 bp bases upstream of PAM, and FrCas9 and conventional SpCas9 have the same range of occurrence of cleavage of the target gene sequence. I understand.
Example 6
This example verified the optimal length of the sgRNA recognition portion in eukaryotic cells of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention by ODN experiments, and the results are shown in FIG. .

(1) Materials: All amino acid sequences, sgRNA sequences and recognized PAMs of Faecalibaculum rodentium edited genes obtained in Examples 1-4 .
(2) Verification method: This example verified the optimal length of the sgRNA recognition portion in eukaryotic cells of the type II CRISPR/Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention by ODN experiments. .
Specific operations are as follows.
(a) A human-derived optimized FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.
(B) 30 bp upstream of GGTA near the target site of SpCas9, which was confirmed in previous studies to have a relatively high cleavage effect, was selected as the target sequence, and an sgRNA with a recognition length of 19 to 23 bp was constructed by the Gibson method, and the sgRNA was A human-derived strong eukaryotic promoter U6 was added upstream to construct a PX330-FrCas9-sgRNA plasmid.
(c) 2.5 μg of PX330-FrCas9-sgRNA plasmid targeting different gene sites constructed above and 1.5 ul of ODN were introduced into HEK293T cells in good condition by electrotransformation, and after 72 hours Whole cells were harvested and DNA extracted.
(d) ODN-PCR is performed using a pair of primers designed near the target gene site and on the ODN sequence, and the presence or absence of a band is observed by agarose gel electrophoresis to rudimentarily confirm the presence or absence of the target cleavage event. , compared the intensity of the bands of sgRNAs with recognition lengths of 19-23 bp.
(e) To quantify the Indel rate of target regions, amplicon high-throughput libraries were prepared to compare the cleavage effects of sgRNAs of recognition length 19-23 bp.
As shown in Figure 7, the optimal recognition length of FrCas9 sgRNA was 21bp, 22bp or 23bp.
Example 7
This example validates the optimal length of the sgRNA backbone in eukaryotic cells of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention by ODN experiments and the results are shown in FIG.
(1) Materials: all amino acid sequences, sgRNA sequences and recognized PAMs of Faecalibaculum rodentium edited genes obtained in Examples 1-6.
(2) Validation method: This example validated the optimal length of sgRNA backbone in eukaryotic cells of the type II CRISPR/Cas9 gene editing system from Faecalibaculum rodentium according to the present invention by ODN experiments.
Specific operations are as follows.
(a) A human-derived optimized FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.
(B) 30 bp upstream of GGTA near the target site of SpCas9, which was confirmed in previous studies to have a relatively high cleavage effect, was selected as the target sequence, and an sgRNA with a backbone length of 71 nt to 95 nt was constructed by the Gibson method, and the sgRNA was A human-derived strong eukaryotic promoter U6 was added upstream to construct a PX330-FrCas9-sgRNA plasmid.
(c) 2.5 μg of the PX330-FrCas9-sgRNA plasmid targeting different gene sites constructed above and 1.5 μl of ODN were introduced into HEK293T cells in good condition by electrotransformation, and after 72 hours Whole cells were harvested and DNA extracted.
(d) ODN-PCR is performed using a pair of primers designed near the target gene site and on the ODN sequence, and the presence or absence of a band is observed by agarose gel electrophoresis to rudimentarily confirm the presence or absence of the target cleavage event. , compared the intensity of the bands of sgRNAs with backbone lengths of 71nt to 95nt.
(e) To quantify the Indel rate of target regions, amplicon high-throughput libraries were prepared to compare the cleavage effects of sgRNAs with backbone lengths from 71nt to 95nt.
As shown in Figures 8 and 3, the optimal sgRNA backbone for FrCas9 was between 83nt and 91nt.
Example 8
In this example, by GUIDE-seq experiments, the cutting effect in eukaryotic cells of the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention is higher than SpCas9, and the off-target rate is higher than SpCas9 verified to be low.
(1) Materials: full amino acid sequence of Faecalibaculum rodentium edited gene obtained in Examples 1-6, 22 bp sgRNA sequence, 5′-GGTA-3′ PAM sequence, SpCas9 amino acid sequence, SpCas9 sgRNA sequence and SpCas9 5′- NGG-3' PAM sequence.
(2) Verification method: In this example, according to the GUIDE-seq experiment, the type II CRISPR / Cas9 gene editing system derived from Faecalibaculum rodentium according to the present invention has a higher cutting effect in eukaryotic cells than SpCas9, and is turned off. It was verified that the target rate is lower than SpCas9.
Specific operations are as follows.
(a) A human-derived optimized FrCas9 protein sequence was synthesized and cloned into the PX330 eukaryotic vector to construct the PX330-FrCas9 plasmid.
(B) Select 30 bp upstream of GGTA near the target site of SpCas9, which was confirmed in previous studies to have a relatively high cleavage effect, as a target sequence, construct an sgRNA with a recognition length of 22 bp by the Gibson method, and upstream of sgRNA A human-derived eukaryotic strong promoter U6 was added to construct a PX330-FrCas9-sgRNA plasmid. At the same time, a 20 bp SpCas9 sgRNA PX330-SpCas9-sgRNA plasmid was constructed.
(c) 2.5 μg of PX330-FrCas9-sgRNA plasmid and PX330-SpCas9-sgRNA plasmid targeting different gene sites constructed above, and 1.5 μl of ODN are electrotransformed into HEK293T cells in good condition 72 hours later, all cells were harvested and DNA was extracted.
(d) ODN-PCR is performed using a pair of primers designed near the target gene site and on the ODN sequence, and the presence or absence of a band is observed by agarose gel electrophoresis to rudimentarily confirm the presence or absence of the target cleavage event. , a GUIDE-seq library was prepared for the DNA for which bands were observed.
(e) Bioinformatic analysis compared the on-target cleavage effect and off-target situation of SpCas9 and FrCas9 at the same site.
As shown in FIG. 9, at the DYRK1A-T2 site, the number of on-target reads for FrCas9 was 3257, which was higher than the number of on-target reads for SpCas9 of 2456. At the same time, in FrCas9, no off-target was detected, whereas in SpCas9, three off-target sites were found. In addition, at the GRIB2B-T9 site, the number of on-target reads for FrCas9 was 34,970, which was higher than the number of on-target reads of 20,434 for SpCas9. At the same time, in FrCas9, no off-target was detected, whereas in SpCas9, three off-target sites were found. From the above data, FrCas9 was found to be a Cas9 protein with better cleavage efficiency and specificity than SpCas9.
Example 9 FrCas9 can be used for base editing (BE).
Many human genetic diseases are due to single nucleotide mutations and cannot be treated by traditional methods. Base editors are one of the newest and most effective ways to achieve precise gene editing. characterized by nicking CRISPR-Cas proteins to locate specific target DNA, modifying and mutating such target DNA using DNA deaminase without double-stranded DNA breaks; Make corrections to the lesion bases. Cytosine base editors (CBE) mutate base pairs C:G to T:A in DNA, and adenine base editors (ABE) mutate base pairs A:T to G:C.
First, three point mutations E796A, H1010A, D1013A were introduced into FrCas9 to produce a different FrCas9 nickase (nFrCas9). E796A nFrCas9 was then ligated into the optimized fourth generation cytosine base editor BE4Gam and the seventh generation adenine base editor ABE7.10. It was observed that the editing window of FrCas9-BE4Gam was 6-10 bases (Fig. 10B) and that of FrCas9-ABE7.10 was 6-8 bases (Fig. 10C). Based on the above characteristics, the target ranges of FrCas9-BE4Gam and FrCas9-ABE7.10 in the ClinVar database were calculated. For pathogenic mutations that could be correctly corrected by FrCas9-BE4Gam, 90.38% (235/260) editing events differed from SpCas9-BE4Gam. For pathogenic mutations that can be correctly corrected by FrCas9-ABE7.10, 92.21% (1196/1297) editing events differed from SpCas9-ABE7.10 (Fig. 10D). Thus, FrCas9 has greatly expanded its targeting in the human genome by TA-rich PAMs and has been used as a base editor to correct mutations associated with human disease.

Also, the PAM of FrCas9(5'-NNTA-3') is palindromic, which allows the sgRNAs to exist in pairs "back to back" (Fig. 10E). This feature expands the range of the FrCas9 base editor by modifying two adjacent editing windows simultaneously (Fig. 10E) and can increase target distribution and density of FrCas9 sgRNAs. The distribution of 5'-GG-3' (representing SpCas9 PAM) and 5'-TA-3' (representing FrCas9 PAM) in the human genome was calculated (Figures 11A and 11B). Compared to SpCas9 (median=5 bp, mean=8.66 bp ) , FrCas9 showed a more dense distribution in the human genome (median=1 bp, mean=6.16 bp), providing additional sites of application. .
Example 10 FrCas9 targets the TATA box to regulate gene expression and can be an effective tool for CRISPR activation and repression.
A TATA box (TATAbox/Hognessbox) is one of elements that constitute a eukaryotic promoter. The matching order is TATA(A/T) A(A/T) (non-template strand sequence). It is located approximately −30 bp (−25 to −32 bp) upstream of the transcription initiation site of many eukaryotic genes and consists essentially of the base pair AT and is the selection that determines the transcription initiation of genes. , is one of the binding sites for RNA polymerase, and transcription is initiated after the RNA polymerase and the TATA box bind tightly. Since the PAM sequence of FrCas9 is a NNTA, it has a natural advantage in targeting the TATA box.
We targeted the TATA box in three genes, ABCA1, UCP3 and RANKL, to test the ability of FrCas9 CRISPR interference (CRISPRi) (Fig. 12A). It involved direct utilization of the active form of FrCas9 to target and cleave the TATA box to disrupt the TATA box. As a result, FrCas9 reduced the expression of ABCA1, UCP3 and RANKL by 31.37%, 49.91% and 39.62%, respectively. In addition, dFrCas9 without cleavage activity was used to bind directly to the TATA box. As a result, the expression of ABCA1, UCP3 and RANKL decreased by 61.67%, 45.61% and 42.60% respectively (Fig. 12B). As such, FrCas9 was found to have unique potential in precisely regulating gene expression.
We also tested the effect of FrCas9 CRISPR activation (CRISPRa) using dFrCas9-VP64, which targets the TATA box directly, and compared its performance to dSpCas9-VP64, which targets the TATA box upstream. These experiments were performed with the ABCA1, SOD1, GH1, BLM2 genes. As a result, it was found that dFrCas9-VP64 can achieve effective transcriptional activation. Also, in ABCA1, GH1, BLM2, the activation fold of dFrCas9-VP64 is higher than dSpCas9-VP64, and in the SOD1 gene, the activation fold of dFrCas9-VP64 is equivalent to dSpCas9-VP64 (Fig. 12C). Therefore, FrCas9 proved to be a promising CRISPR screening tool due to its unique 5'-NNTA-3'PAM.
Example 11 FrCas9 can be used for precision gene editing (Prime Editing, PE).
Prime Editing (PE) is a completely new precision gene editing tool. This technology can achieve free substitution of single nucleotides and accurate insertion/deletion of multiple nucleotides, greatly reducing harmful by-products generated by indels during gene editing, significantly improving editing accuracy, It is considered to be a major advance in gene editing that has the potential to overcome fundamental obstacles to treating genetic diseases with existing gene-editing methods. The PE system contains two parts, a modified guide RNA (pegRNA) and a prime editor protein. The pegRNA has a dual function, being able to direct the editing protein to the target site as well as containing the template sequence for editing. The prime editor protein is a fusion of a mutated Cas9 nickase (which can cut only one strand of DNA) and reverse transcriptase. After cleaving the target site with Cas9, the reverse transcriptase reverse transcribes the pegRNA as a template, thereby incorporating the necessary edits into the DNA strand, the modified sequences preferentially displacing the original genomic DNA, and the target A permanent edit was made to the DNA at the position (Fig. 13).
Based on the biological properties of FrCas9, fuse FrCas9 with reverse transcriptase, design corresponding pegRNA based on its PAM sequence, construct FrCas9-PE gene editing system, and optimize its gene editing performance did. The pegRNA designed based on the FrCas9 PAM sequence had two sequences added to the 3' end sequence of the pegRNA compared to the sgRNA. The first sequence is a primer binding site (PBS), capable of complementary binding to the ends of the cleaved target DNA strand, thereby initiating the reverse transcription process. The second sequence is a reverse transcription template (RT template) with targeted point mutations or insertion-deletion mutations to achieve precise gene editing. Previous studies have shown that the sequence lengths of PBS and RT templates significantly affected the gene editing efficiency of the PE system and differed by gene site. Therefore, optimization of the editing efficiency was investigated by combining different lengths of PBS and RT template sequences.
At the cellular level, the gene-editing effects of the FrCas9-PE system were rudimentary verified. In HEK293T cells, we verified the gene-editing function of the FrCas9-PE system by targeting the HEK293T-RNF2 gene site, which is often used to evaluate the gene-editing efficiency of CRISPR. Current experimental results indicated that FrCas9-PE can exert a base-editing effect at specific sites. After that, the existing FrCas9-PE was further modified such as codon optimization, optimization of the position and number of nuclear localization sequences, and reverse transcriptase modification. Finally, a novel FrCas9-PE gene-editing system with high base-editing efficiency, low off-target reactions, and low gene-editing by-products (indel) was developed (FIG. 14).

Claims (11)

A type II CRISPR/Cas9 gene editing system,
Cas9 protein, accessory protein, crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA), including Cas9 protein and guide RNA formed by binding tracrRNA to crRNA RNP complex (ribonucleoprotein complex) functions in the form of
The Cas9 protein is a DNA endonuclease, and the Cas9 protein is the amino acid sequence shown in SEQ ID NO: 1, or at least 80%, 85%, 90%, 95%, 98% of the amino acid sequence shown in SEQ ID NO: 1 %, or 99% amino acid sequence homology,
A type II CRISPR/Cas9 gene editing system characterized by: said Cas9 protein cleaves double-stranded DNA complementary to crRNA upstream of the PAM sequence with a nuclease domain, said nuclease domain being an HNH-like nuclease domain and/or a RuvC-like nuclease domain;
The type II CRISPR/Cas9 gene editing system according to claim 1, characterized in that: The accessory proteins include Cas1 accessory protein, Cas2 accessory protein and Csn2 accessory protein,
The Cas1 accessory protein is the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence set forth in SEQ ID NO:2 has an array,
The Cas2 accessory protein is the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 3 has an array,
The Csn2 accessory protein is the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence set forth in SEQ ID NO: 4 having an array,
The type II CRISPR/Cas9 gene editing system according to claim 1, characterized in that: The CRISPR RNA is generated by transcription from a CRISPR array, and the CRISPR RNA is at least 80%, 85%, 90%, 95% of the RNA sequence set forth in SEQ ID NO:5 or the nucleic acid sequence set forth in SEQ ID NO:5. , 98% or 99% homology, wherein the CRISPR array comprises a direct repeat sequence and a spacer sequence, wherein the direct repeat sequence is the nucleic acid sequence shown in SEQ ID NO: 6, or the sequence a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% homology with the nucleic acid sequence shown in number 6, wherein the spacer sequence is the nucleic acid 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 shown in SEQ ID NO:7;
The type II CRISPR/Cas9 gene editing system according to claim 1, characterized in that: The transactivating CRISPR RNA contains a sequence complementary to a direct repeat sequence of CRISPR RNA, and the transactivating CRISPR RNA is the nucleic acid sequence shown in SEQ ID NO: 8, or the nucleic acid shown in SEQ ID NO: 8 having a nucleic acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% homologous to the sequence;
The type II CRISPR/Cas9 gene editing system according to claim 1, characterized in that: The scaffold of the guide RNA, which is crRNA bound to tracrRNA, is composed of a sequence of 7 to 24 nt of the crRNA direct repeat sequence and the tracrRNA sequence, and these two parts are "GAAA", "TGAA", and "AAAC". can be fused via a linker to form an sgRNA backbone,
Preferably, the sgRNA backbone formed by fusing a 20 nt direct repeat sequence and the full-length sequence of tracrRNA via "GAAA" to form is shown in SEQ ID NO: 9, more preferably the high efficiency variant of this sgRNA backbone is ,
an sgRNA backbone formed by fusing the first 18-14 nt of the direct repeat sequence of the crRNA and the last 69-65 nt of the tracrRNA through a linker sequence (such as "GAAA"), which further comprises
(a) a backbone represented by SEQ ID NO: 10, which has a length of 91 nt and contains a direct repeat sequence of 18 nt and a tracrRNA of 69 nt;
(b) an sgRNA backbone having a length of 89 nt, containing a direct repeat sequence of 17 nt and a tracrRNA of 68 nt, and represented by SEQ ID NO: 11;
(c) an sgRNA backbone of 87 nt in length, containing a 16 nt direct repeat sequence and 67 nt of tracrRNA, and represented by SEQ ID NO: 12;
(d) an sgRNA backbone having a length of 85 nt, containing a direct repeat sequence of 15 nt and a tracrRNA of 66 nt, and represented by SEQ ID NO: 13;
(e) a backbone represented by SEQ ID NO: 14, which has a length of 83 nt and contains a direct repeat sequence of 14 nt and a tracrRNA of 65 nt;
A skeleton selected from the group consisting of
said sgRNA backbone may have a nucleic acid sequence with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% homology to any one of SEQ ID NOS: 9-14;
The type II CRISPR/Cas9 gene editing system according to claim 1, characterized in that: The type II CRISPR / Cas9 gene editing system is derived from Faecalibaculum rodentium, binding or cleaving specific DNA in gene editing, binding or cleaving the specific DNA, the guide RNA is specific The length range of the binding portion paired with the guide RNA and the specific DNA target site is 14 to 30 bp. can be,
the specific DNA is prokaryotic or eukaryotic DNA;
The type II CRISPR/Cas9 gene editing system according to claim 1, characterized in that: The length of the paired binding portion between the guide RNA and the specific DNA target site is 21 bp, 22 bp or 23 bp, wherein the RNP protein complex comprises a 14 bp very sensitive to base mismatches, said 14 bp being the seed region;
The type II CRISPR/Cas9 gene editing system according to claim 7, characterized in that: The protospacer-adjacent motif sequence required for the DNA binding or cleaving function is 5'-NNTA-3' downstream of the guide RNA/sgRNA recognition sequence, where N is A, T, C or G. be,
The type II CRISPR/Cas9 gene editing system according to claim 7, characterized in that: CRISPR/Cas9 gene editing system for editing or joining DNA, activating CRISPR or interfering with CRISPR, wherein the CRISPR/Cas9 gene editing system is II according to any one of claims 1 to 9 A type CRISPR/Cas9 gene editing system,
A CRISPR/Cas9 gene editing system characterized by: A CRISPR / Cas9 gene editing system for the production of nickase, inactive Cas9, Base Editor or Prime Editor, wherein the CRISPR / Cas9 gene editing system is according to claims 1 to 9 A type II CRISPR/Cas9 gene editing system according to any one of
A CRISPR/Cas9 gene editing system characterized by: