CN112111471A - FnCpf1 mutant for identifying PAM sequence in broad spectrum and application thereof - Google Patents
FnCpf1 mutant for identifying PAM sequence in broad spectrum and application thereof Download PDFInfo
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Abstract
The invention provides a CRISPR nuclease FnCpf1 mutant, which is characterized in that the following mutations exist relative to a wild type FnCpf 1: K671R/E566V/D751V/N508V/N637V, K671V/E566V/D751V/F570V/N634V/R755V, K671V/E566V/D751V/S518V/K639V, K671V/E566V/D751V/F570V/E686V, K671V/E566V/D751V/K613V/N534V/G664V, K671V/E566V/D751V/D751V/N637V, K671V/E566/D V/D664V/K V/Y36613/D V/Y751V/F751/K V/K671V/K671/K V/K671V/K V/L751/L V/L36724/L V/L36. Proved by experiments, the coding gene of the mutant is used for gene editing, has higher editing efficiency and wider editing range than wild FnCpf1, and has higher application value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a CRISPR nuclease FnCpf1 mutant and application thereof.
Background
With the development of CRISPR-Cas system, it is widely used for gene editing of various species, and it has now become a powerful genome editing tool for inserting, deleting or modifying genome sequences of organisms. In order to improve the accuracy and editing efficiency of single-point mutation, a single-base editing system combining CRISPR-Cas protein and cytosine/adenine deaminase is a new generation of more precise gene editing tool developed recently. The single Base Editing (BE) technique can switch from one Base pair to another (C/G-T/A or A/T-G/C) accurately and irreversibly without causing DNA double strand breaks and homologous recombination. Cpf1 acts as another CRISPR nuclease in addition to Cas9, whose RNA endonuclease activity makes it potentially advantageous in multiple gene targeting. The ability of Cpf1 to manipulate multiple genes simultaneously means that it can implement a more complex genome regulatory network at the system level. In addition, the Cpf1 system has the characteristics of simpler crRNA (about 40nt), smaller molecular weight, low off-target rate and the like. Thus, Cpf1(dCpf1) with lost cleavage activity still has great potential for multigene regulation, multi-target base editing.
Although the Cpf1 enzyme has shown great advantages in multi-target gene editing and transcriptional regulation, its demand for 5' -TTTV/TTV PAM has limited its development and application of gene editing technology in GC-rich species. In the work reported so far, mutants RVR and RR of AsCpf1 were designed to recognize TATV and TYCV/CCCC PAMs, respectively (Linyi Gao, David B T Cox, Winston X Yan. et al. engineered Cpf1 variants with altered PAM specificities. Nat Biotechnology.35, 789-792 (2017.);
the enAsCas12a (E174R/S542R/K548R) further extended the recognition range of PAMs to TTYN/VTTV/TRTV (Benjamin P. Kleinsight, Alexander A. Sousa, Russell T. Walton1.et al. engineered CRISPR-Cas12a variants with engineered activities and expressed targeting sequences for gene, epigene and base editing. Nature Biotechnology 37, 276-282 (2019)). Furthermore, the extension of the recognition range of PAMs by mutation of FnCpf1 was partially studied, but this work neglected the effect at position-4 in the PAM sequence (lipid Wang, Haojun Wang. et. improved CRISPR-Cas12 a-associated one-pot DNA editing. Biotechnology and bioengineering.2019; 116: 1463. 1474.). To date, Cpf1 has not been recognized to bind to most PAMs sequences, particularly GC-rich PAMs sequences.
Therefore, there is a great need to develop Cpf1 mutants that can recognize more GC-rich PAMs sequences (preferably broad spectrum) to improve their application range and flexibility.
Disclosure of Invention
Aiming at the defects in the prior art, the inventor constructs an directed evolution system in Escherichia coli to screen Cpf1 mutants capable of identifying more GC-rich PAMs to improve the application range and flexibility.
The invention provides a CRISPR nuclease FnCpf1 mutant, which has the following mutations relative to a wild type FnCpf1 of an amino acid sequence shown as SEQ ID NO. 2: K671R/E566V/D751V/N508V/N637V, K671V/E566V/D751V/F570V/N634V/R755V, K671V/E566V/D751V/S518V/K639V, K671V/E566V/D751V/F570V/E686V, K671V/E566V/D751V/K613V/N534V/G664V, K671V/E566V/D751V/D751V/N637V, K671V/E566/D V/D664V/K V/Y36613/D V/K751V/K671V/K671/K V/L751/L V/L36724/L V.
Preferably, the mutant has the following mutations relative to wild-type FnCpf 1:
K671R/E566V/D751G/K613N/Y724C/F570L/R690I/L662I、K671R/E566V/D751G/K613N/N637S/N534K/G664V、K671R/E566V/D751G/K613N/F570L/G664S/N637Y、K671R/E566V/D751G/K613N/Y724C/F570L。
most preferably, the mutation is relative to wild-type FnCpf1, which has the following mutations: K671R/E566V/D751G/K613N/Y724C/F570L/R690I/L662I.
The present invention further provides a gene encoding the mutant as described above. The nucleotide sequence of the gene is preferably shown as SEQ ID NO. 4.
The invention also provides a vector containing the gene as described, preferably a vector for gene editing.
Further provided is a recombinant cell line containing the gene, preferably a recombinant cell line containing the above-mentioned vector, such as E.coli and the like.
The invention also provides the application of the gene in gene editing, such as gene editing of bacterial genome.
The above mutants of the present invention show higher recognition and binding ability at sites of atypical PAMs compared to wild-type FnCpf1, while still retaining potent activity on typical TTTV PAMs. By designing a cytosine base editor aiming at the mutant, the base editor of the mutant is proved to have higher editing efficiency and wider editing range in escherichia coli than a wild type, and the mutation has obvious advantages in the PAM broad spectrum.
Drawings
FIG. 1 is a schematic diagram of the gene circuit of interference suppression of YFP fluorescence by dCpf 1. In this loop, dFnCpf1 expression was induced by IPTG, and constitutive promoters J23119 and J23100 expressed crRNA and YFP constantly. Under IPTG induction, dFnCpf1 protein is combined with crRNA to form a complex, a PAM sequence upstream of YFP is identified and combined to a target DNA so as to inhibit the expression of reporter gene YFP, so that the identification and combination efficiency between dCpf1 mutant and PAM sequence can be quantified through YFP fluorescence value inhibition multiple.
Figure 2. flow chart of directed evolution of CRISPR nuclease dFnCpf 1. The directed evolution process is to construct a mutant library with dFnCpf1 by using error-prone PCR, screen bacteria with obviously reduced YFP fluorescence value by using flow cytometry sorting, further correct the inhibition efficiency by using a flow cytometer, sequence, screen the best mutant and screen the next round of evolutionary mutation.
FIG. 3 PAM preference profile of wild type dFnCpf1 and 8 selected mutants. The most efficient mutants selected from the 8 evolutionary pathways were evaluated for their preference for 64 PAMs (NNNV, V not included) and compared to wild-type dFnCpf 1. dFnCpf1(VRG/N508H/N637S), dFnCpf1(VRG/F570L/N634D/R755K), dFnCpf1(VRG/S518G/K639R), dFnCpf1(VRG/F570L/E686D) were selected from mutants obtained by directed evolution that recognized PAM GCCG, CCGC, GCGC and CGCC, respectively. dFnCpf1(VRGN/N637S/N534K/G664V), dFnCpf1(VRGN/F570L/G664S/N637Y), and dFnCpf1(VRGN/Y724C/F570L/R690I/L662I) were selected from mutants obtained by directed evolution that recognized PAM CGCC, GGCC, CGGC and GGGC (SGSC), respectively. VRG represents the mutation sites E566V, K671R and D751G, and N represents the mutation site K613N. The YFP fluorescence intensity after 200 μ M IPTG induction was taken as the characterization value of the PAM preference profile.
FIG. 4 shows the base-editing efficiency of cytosine base editor dFnCpf1-BE/dbsFnCpf1-BE on a target DNA sequence with a PAM sequence of TTTC or non-TTTC, respectively, i.e., the base substitution rate of C to T in the target site, under the induction of IPTG. The experimental data are obtained by statistics of the second generation sequencing results.
Detailed Description
The invention will be further illustrated by the following specific research procedures and embodiments in order to better understand the invention, but not to be construed as being limited thereto.
FnCpf1 from Francisella novicida was chosen for directed evolution, targeting positions-2 to-4 (-1 base with great selectivity and therefore not considered) of the PAM sequence, in an attempt to extend the preference of the PAM sequence to GC-rich PAMs. The following is the FnCpf1 nucleotide sequence (SEQ ID NO: 1).
ATGTCAATTTATCAAGAATTTGTTAATAAATATAGTTTAAGTAAAACTCTAAGATTTGAGTTAATCCCACAGGGTAAAACACTTGAAAACATAAAAGCAAGAGGTTTGATTTTAGATGATGAGAAAAGAGCTAAAGACTACAAAAAGGCTAAACAAATAATTGATAAATATCATCAGTTTTTTATAGAGGAGATATTAAGTTCGGTTTGTATTAGCGAAGATTTATTACAAAACTATTCTGATGTTTATTTTAAACTTAAAAAGAGTGATGATGATAATCTACAAAAAGATTTTAAAAGTGCAAAAGATACGATAAAGAAACAAATATCTGAATATATAAAGGACTCAGAGAAATTTAAGAATTTGTTTAATCAAAACCTTATCGATGCTAAAAAAGGGCAAGAGTCAGATTTAATTCTATGGCTAAAGCAATCTAAGGATAATGGTATAGAACTATTTAAAGCCAATAGTGATATCACAGATATAGATGAGGCGTTAGAAATAATCAAATCTTTTAAAGGTTGGACAACTTATTTTAAGGGTTTTCATGAAAATAGAAAAAATGTTTATAGTAGCAATGATATTCCTACATCTATTATTTATAGGATAGTAGATGATAATTTGCCTAAATTTCTAGAAAATAAAGCTAAGTATGAGAGTTTAAAAGACAAAGCTCCAGAAGCTATAAACTATGAACAAATTAAAAAAGATTTGGCAGAAGAGCTAACCTTTGATATTGACTACAAAACATCTGAAGTTAATCAAAGAGTTTTTTCACTTGATGAAGTTTTTGAGATAGCAAACTTTAATAATTATCTAAATCAAAGTGGTATTACTAAATTTAATACTATTATTGGTGGTAAATTTGTAAATGGTGAAAATACAAAGAGAAAAGGTATAAATGAATATATAAATCTATACTCACAGCAAATAAATGATAAAACACTCAAAAAATATAAAATGAGTGTTTTATTTAAGCAAATTTTAAGTGATACAGAATCTAAATCTTTTGTAATTGATAAGTTAGAAGATGATAGTGATGTAGTTACAACGATGCAAAGTTTTTATGAGCAAATAGCAGCTTTTAAAACAGTAGAAGAAAAATCTATTAAAGAAACACTATCTTTATTATTTGATGATTTAAAAGCTCAAAAACTTGATTTGAGTAAAATTTATTTTAAAAATGATAAATCTCTTACTGATCTATCACAACAAGTTTTTGATGATTATAGTGTTATTGGTACAGCGGTACTAGAATATATAACTCAACAAATAGCACCTAAAAATCTTGATAACCCTAGTAAGAAAGAGCAAGAATTAATAGCCAAAAAAACTGAAAAAGCAAAATACTTATCTCTAGAAACTATAAAGCTTGCCTTAGAAGAATTTAATAAGCATAGAGATATAGATAAACAGTGTAGGTTTGAAGAAATACTTGCAAACTTTGCGGCTATTCCGATGATATTTGATGAAATAGCTCAAAACAAAGACAATTTGGCACAGATATCTATCAAATATCAAAATCAAGGTAAAAAAGACCTACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCTATCAAGGATCTTTTAGATCAAACTAATAATCTCTTACATAAACTAAAAATATTTCATATTAGTCAGTCAGAAGATAAGGCAAATATTTTAGACAAGGATGAGCATTTTTATCTAGTATTTGAGGAGTGCTACTTTGAGCTAGCGAATATAGTGCCTCTTTATAACAAAATTAGAAACTATATAACTCAAAAGCCATATAGTGATGAGAAATTTAAGCTCAATTTTGAGAACTCGACTTTGGCTAATGGTTGGGATAAAAATAAAGAGCCTGACAATACGGCAATTTTATTTATCAAAGATGATAAATATTATCTGGGTGTGATGAATAAGAAAAATAACAAAATATTTGATGATAAAGCTATCAAAGAAAATAAAGGCGAGGGTTATAAAAAAATTGTTTATAAACTTTTACCTGGCGCAAATAAAATGTTACCTAAGGTTTTCTTTTCTGCTAAATCTATAAAATTTTATAATCCTAGTGAAGATATACTTAGAATAAGAAATCATTCCACACATACAAAAAATGGTAGTCCTCAAAAAGGATATGAAAAATTTGAGTTTAATATTGAAGATTGCCGAAAATTTATAGATTTTTATAAACAGTCTATAAGTAAGCATCCGGAGTGGAAAGATTTTGGATTTAGATTTTCTGATACTCAAAGATATAATTCTATAGATGAATTTTATAGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATATCAGAGAGCTATATTGATAGCGTAGTTAATCAGGGTAAATTGTACCTATTCCAAATCTATAATAAAGATTTTTCAGCTTATAGCAAAGGGCGACCAAATCTACATACTTTATATTGGAAAGCGCTGTTTGATGAGAGAAATCTTCAAGATGTGGTTTATAAGCTAAATGGTGAGGCAGAGCTTTTTTATCGTAAACAATCAATACCTAAAAAAATCACTCACCCAGCTAAAGAGGCAATAGCTAATAAAAACAAAGATAATCCTAAAAAAGAGAGTGTTTTTGAATATGATTTAATCAAAGATAAACGCTTTACTGAAGATAAGTTTTTCTTTCACTGTCCTATTACAATCAATTTTAAATCTAGTGGAGCTAATAAGTTTAATGATGAAATCAATTTATTGCTAAAAGAAAAAGCAAATGATGTTCATATATTAAGTATAGACAGAGGTGAAAGACATTTAGCTTACTATACTTTGGTAGATGGTAAAGGCAATATCATCAAACAAGATACTTTCAACATCATTGGTAATGATAGAATGAAAACAAACTACCATGATAAGCTTGCTGCAATAGAGAAAGATAGGGATTCAGCTAGGAAAGACTGGAAAAAGATAAATAACATCAAAGAGATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAAATAGCTAAGCTAGTTATAGAGTATAATGCTATTGTGGTTTTTGAGGATTTAAATTTTGGATTTAAAAGAGGGCGTTTCAAGGTAGAGAAGCAGGTCTATCAAAAGTTAGAAAAAATGCTAATTGAGAAACTAAACTATCTAGTTTTCAAAGATAATGAGTTTGATAAAACTGGGGGAGTGCTTAGAGCTTATCAGCTAACAGCACCTTTTGAGACTTTTAAAAAGATGGGTAAACAAACAGGTATTATCTACTATGTACCAGCTGGTTTTACTTCAAAAATTTGTCCTGTAACTGGTTTTGTAAATCAGTTATATCCTAAGTATGAAAGTGTCAGCAAATCTCAAGAGTTCTTTAGTAAGTTTGACAAGATTTGTTATAACCTTGATAAGGGCTATTTTGAGTTTAGTTTTGATTATAAAAACTTTGGTGACAAGGCTGCCAAAGGCAAGTGGACTATAGCTAGCTTTGGGAGTAGATTGATTAACTTTAGAAATTCAGATAAAAATCATAATTGGGATACTCGAGAAGTTTATCCAACTAAAGAGTTGGAGAAATTGCTAAAAGATTATTCTATCGAATATGGGCATGGCGAATGTATCAAAGCAGCTATTTGCGGTGAGAGCGACAAAAAGTTTTTTGCTAAGCTAACTAGTGTCCTAAATACTATCTTACAAATGCGTAACTCAAAAACAGGTACTGAGTTAGATTATCTAATTTCACCAGTAGCAGATGTAAATGGCAATTTCTTTGATTCGCGACAGGCGCCAAAAAATATGCCTCAAGATGCTGATGCCAATGGTGCTTATCATATTGGGCTAAAAGGTCTGATGCTACTAGGTAGGATCAAAAATAATCAAGAGGGCAAAAAACTCAATTTGGTTATCAAAAATGAAGAGTATTTTGAGTTCGTGCAGAATAGGAATAACTAG。
Wherein 750bp of the middle part was used as an error-prone PCR sequence (SEQ ID NO: 3):
GGTAAAAAAGACCTACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCTATCAAGGATCTTTTAGATCAAACTAATAATCTCTTACATAAACTAAAAATATTTCATATTAGTCAGTCAGAAGATAAGGCAAATATTTTAGACAAGGATGAGCATTTTTATCTAGTATTTGAGGAGTGCTACTTTGAGCTAGCGAATATAGTGCCTCTTTATAACAAAATTAGAAACTATATAACTCAAAAGCCATATAGTGATGAGAAATTTAAGCTCAATTTTGAGAACTCGACTTTGGCTAATGGTTGGGATAAAAATAAAGAGCCTGACAATACGGCAATTTTATTTATCAAAGATGATAAATATTATCTGGGTGTGATGAATAAGAAAAATAACAAAATATTTGATGATAAAGCTATCAAAGAAAATAAAGGCGAGGGTTATAAAAAAATTGTTTATAAACTTTTACCTGGCGCAAATAAAATGTTACCTAAGGTTTTCTTTTCTGCTAAATCTATAAAATTTTATAATCCTAGTGAAGATATACTTAGAATAAGAAATCATTCCACACATACAAAAAATGGTAGTCCTCAAAAAGGATATGAAAAATTTGAGTTTAATATTGAAGATTGCCGAAAATTTATAGATTTTTATAAACAGTCTATAAGTAAGCATCCGGAGTGGAAAGATTTTGGATTTAGATTTTCTGATACTCAAAGATATAATTCTATAGATGAATTTTATAGAGAAGTTGAAAAT。
the number of the encoded amino acid residues is 1300, and the specific amino acid sequence (SEQ ID NO: 2) is as follows: MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN are provided.
Example one
First, a test screening system for interference suppression of YFP fluorescence value by dCpf1 was constructed in e.coli to detect its transcription suppression (fig. 1). Plasmids created in this experiment were all constructed using the Gibson assembly method or the Golden Gate assembly method and verified by Sanger sequencing. The 750bp DNA sequence on selected FnCpf1 was subjected to error-prone PCR to construct a mutation library and inserted into a vector containing the pTac inducible promoter, the p15A origin of replication and the ampicillin selection marker by the Golden Gate method using the BsaI cleavage site. The vector is used for controlling the inducible expression of the dCpf1 enzyme and mutants thereof. The crRNA plasmid contains a constitutive promoter (J23119), ColE1 origin of replication, and a chloramphenicol selection marker for expression of sufficient crRNA. The reporter plasmid contained the YFP reporter gene, promoter J23100, pSC101 origin of replication, and kanamycin selectable marker.
The test screening system can systematically quantify the binding inhibition efficiency of dCpf1 mutant and is useful for subsequent high-throughput screening. Based on the sequence and structure homology of Cpf1 family enzymes, a homologous mutant dFnCpf1(N607R/K671R) of dAsCpf1(G532R/K595R) is found to have certain capability of recognizing PAM CCCC. Therefore, the mixed dFnCpf1 mutants N607R, K671R and N607R/K671R were used as templates, and 750bp DNA sequence (SEQ ID NO: 2) containing PI functional domain was selected for error-prone PCR and assembled by Golden Gate to construct dFnCpf1 mutant library. Second, a constructed test screening system of dCpf1 interference suppression of YFP fluorescence values was used to identify dCpf1 mutants that recognize GC-rich PAMs sequences. Under IPTG induction, the dFnCpf1 protein mutant is combined with crRNA to form a complex, recognizes an SSSC PAM (S represents a base C or G) sequence at the upstream of YFP and is combined to a target DNA so as to reduce the fluorescence value of YFP. The invention screens protein mutants that can recognize different GC-rich PAM sequences by applying artificial selection pressure.
In this, mutants were screened by flow cytometry for fluorescence (FIG. 2). The specific process is as follows:
coli strain DH5a was used for this experiment. LB medium (10g/L trypsin, 5g/L yeast extract, 10g/L NaCl) was used as growth medium for E.coli. M9 Medium (12.8g/L Na)2HPO4·7H2O、3g/L KH2PO4、0.5g/L NaCl、1.67g/L NH4Cl, 1mM thiamine hydrochloride, 0.4% glucose, 0.2% tyrosine, 2mM MgSO4、0.1mM CaCl2) Used as a medium for flow cytometry for fluorometric analysis of cells.
The transformed Escherichia coli strain is cultured in LB culture medium overnight, the bacterial cells are added into M9 culture medium containing corresponding antibiotic to be diluted 196 times, cultured for 3h at 37 ℃, added into M9 culture medium containing corresponding antibiotic and 200 mu M IPTG to be diluted 1000 times, and shaken for 8h at 37 ℃. Before flow cytometry analysis, bacterial cells were diluted with PBS containing 2mg/ml kanamycin to stop protein expression. Fluorescence intensity of YFP was measured using Calibur flow cytometer (BD Biosciences, CA, USA) and appropriate parameter values (FSC 440, SSC 260, FITC 480) were set. At least 50,000 cells per sample were collected and the geometric mean of fluorescence intensity for each sample was analyzed using FlowJo software. Before mutation screening, the strains were cultured overnight (about 14 hours), induced for 6 hours after dilution with M9 medium containing 200. mu.M IPTG, and then sorted into fresh LB medium using a BD in-flow cell sorter (BD, USA) for cells with relatively low fluorescence (below the threshold set for the positive control, i.e., the lowest fluorescence of the positive control). After 3 hours of cell recovery, the sorted cells were placed on LB agar, and the monoclonals were picked and flow cytometric verified (BD Fortessa, USA). Cells with relatively low fluorescence were sequenced and collected for the next mutant screen. Positive controls (DH5a containing plasmid pSC101-J23100-yfp) and negative controls (DH5a containing plasmid pSC101-J23100, pCole1-J23119-crRNA and p15a-ptacc-dFncpf1) were used to set the corresponding fluorescence channel gains.
Different efficient mutants were evolved based on 8 different GC-rich PAMs (SSSC, S ═ G, C). As can be seen from the experimental results, 86 PAM sequences were obtained that effectively recognized the corresponding GC-rich resulting in a significant reduction in YFP fluorescence values. Wild type dFnCpf1 has no recognition capability on SSSC PAMs, while the dFnCpf1 mutant with extremely strong recognition capability on PAM CCCC and PAM CCGC is obtained in the experiment, and the inhibition multiple on YFP fluorescence value can reach more than 100 times (equivalent to the recognition capability of wild type dFnCpf1 on PAM TTTC); the dFnCpf1 mutant with stronger identification capability on PAM GCCG and PAM GCGC has the inhibition multiple of YFP fluorescence value up to 60-70 times; mutants with obvious identification capability on PAM CGCC, GGCC, CGGC and GGGC have inhibition times of more than 30 times on YFP fluorescence value. Specific results are shown in table 1.
TABLE 1 efficient dFnCpf1 mutant obtained by directed evolution screening
Example two
1. PAM preference spectrum analysis
To obtain mutants that could provide more PAM selectivity, the 8 most potent dFnCpf1 mutants were selected in each of the 8 PAM mutation pathways for a full spectrum preference test of 64 PAMs (NNNV, V not included) and compared to the wild-type dFnCpf 1. The construction method adopts inverse PCR and Gibson connection to obtain 64 reporter gene vectors containing different PAM (NNNC) sequences at the upstream of YFP. 64 PAM plasmids were transformed into E.coli DH5a containing plasmid of dFnCpf1 mutant and crRNA plasmid, respectively. The fluorescence intensity of YFP was then measured using flow cytometry and the data was analyzed using FlowJo software. The preference curves for PAM were analyzed and shown by Matlab.
From the results (see fig. 3), wild-type dFnCpf1 was most active in NTTV PAMs, especially TTTV PAM. In contrast, the dFnCpf1 mutants capable of recognizing SCSC PAMs (VRG/N508H/N637S, VRG/F570L/N634D/R755K, VRG/S518G/K639R, VRG/F570L/E686D) recognized the highest ability in the PAMs NCCV and NCTV, respectively, whereas the wild-type dFnCpf1 could not recognize these PAMs substantially. From the PAM preference profile, it can be seen that these four mutants have a significant improvement in PAM broad-spectrum compared to the wild type. Most importantly, it was found that mutants capable of recognizing SGSC PAMs (VRGN/N637S/N534K/G664V, VRGN/F570L/G664S/N637Y, VRGN/Y724C/F570L, VRGN/Y724C/F570L/R690I/L662I) could recognize almost all 64 PAMs, especially GC-rich PAMs. Defining the ' YFP fluorescence value <200 after induction as ' effective recognition ', the effective recognition efficiency of dFnCpf1(VRGN/Y724C/F570L/R690I/L662I) is found to reach 81.3% (52/64). In addition, the mutant was able to efficiently recognize 90% (27/30) of the GC-rich PAMs sequences (two or more C/G in the PAM sequence) (table 2). Therefore, the mutant dFnCpf1(VRGN/Y724C/F570L/R690I/L662I) with the most improved broad spectrum is named dbsFnCpf1, and the recognizable PAM sequence is nearly 10 times enlarged compared with the wild type FnCpf1, so that the regulation range is greatly improved. The PAM sequences recognizable by the other three mutants capable of recognizing SGSC PAMs (VRGN/N637S/N534K/G664V, VRGN/F570L/G664S/N637Y and VRGN/Y724C/F570L) are also enlarged by more than 5 times compared with the wild-type dFnCpf 1.
Wherein the nucleotide sequence (SEQ ID NO: 4) of the dbsFnCpf1 gene is as follows:
ATGTCAATTTATCAAGAATTTGTTAATAAATATAGTTTAAGTAAAACTCTAAGATTTGAGTTAATCCCACAGGGTAAAACACTTGAAAACATAAAAGCAAGAGGTTTGATTTTAGATGATGAGAAAAGAGCTAAAGACTACAAAAAGGCTAAACAAATAATTGATAAATATCATCAGTTTTTTATAGAGGAGATATTAAGTTCGGTTTGTATTAGCGAAGATTTATTACAAAACTATTCTGATGTTTATTTTAAACTTAAAAAGAGTGATGATGATAATCTACAAAAAGATTTTAAAAGTGCAAAAGATACGATAAAGAAACAAATATCTGAATATATAAAGGACTCAGAGAAATTTAAGAATTTGTTTAATCAAAACCTTATCGATGCTAAAAAAGGGCAAGAGTCAGATTTAATTCTATGGCTAAAGCAATCTAAGGATAATGGTATAGAACTATTTAAAGCCAATAGTGATATCACAGATATAGATGAGGCGTTAGAAATAATCAAATCTTTTAAAGGTTGGACAACTTATTTTAAGGGTTTTCATGAAAATAGAAAAAATGTTTATAGTAGCAATGATATTCCTACATCTATTATTTATAGGATAGTAGATGATAATTTGCCTAAATTTCTAGAAAATAAAGCTAAGTATGAGAGTTTAAAAGACAAAGCTCCAGAAGCTATAAACTATGAACAAATTAAAAAAGATTTGGCAGAAGAGCTAACCTTTGATATTGACTACAAAACATCTGAAGTTAATCAAAGAGTTTTTTCACTTGATGAAGTTTTTGAGATAGCAAACTTTAATAATTATCTAAATCAAAGTGGTATTACTAAATTTAATACTATTATTGGTGGTAAATTTGTAAATGGTGAAAATACAAAGAGAAAAGGTATAAATGAATATATAAATCTATACTCACAGCAAATAAATGATAAAACACTCAAAAAATATAAAATGAGTGTTTTATTTAAGCAAATTTTAAGTGATACAGAATCTAAATCTTTTGTAATTGATAAGTTAGAAGATGATAGTGATGTAGTTACAACGATGCAAAGTTTTTATGAGCAAATAGCAGCTTTTAAAACAGTAGAAGAAAAATCTATTAAAGAAACACTATCTTTATTATTTGATGATTTAAAAGCTCAAAAACTTGATTTGAGTAAAATTTATTTTAAAAATGATAAATCTCTTACTGATCTATCACAACAAGTTTTTGATGATTATAGTGTTATTGGTACAGCGGTACTAGAATATATAACTCAACAAATAGCACCTAAAAATCTTGATAACCCTAGTAAGAAAGAGCAAGAATTAATAGCCAAAAAAACTGAAAAAGCAAAATACTTATCTCTAGAAACTATAAAGCTTGCCTTAGAAGAATTTAATAAGCATAGAGATATAGATAAACAGTGTAGGTTTGAAGAAATACTTGCAAACTTTGCGGCTATTCCGATGATATTTGATGAAATAGCTCAAAACAAAGACAATTTGGCACAGATATCTATCAAATATCAAAATCAAGGTAAAAAAGACCTACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCTATCAAGGATCTTTTAGATCAAACTAATAATCTCTTACATAAACTAAAAATATTTCATATTAGTCAGTCAGAAGATAAGGCAAATATTTTAGACAAGGATGAGCATTTTTATCTAGTATTTGTGGAGTGCTACCTTGAGCTAGCGAATATAGTGCCTCTTTATAACAAAATTAGAAACTATATAACTCAAAAGCCATATAGTGATGAGAAATTTAAGCTCAATTTTGAGAACTCGACTTTGGCTAATGGTTGGGATAAAAATAATGAGCCTGACAATACGGCAATTTTATTTATCAAAGATGATAAATATTATCTGGGTGTGATGAATAAGAAAAATAACAAAATATTTGATGATAAAGCTATCAAAGAAAATAAAGGCGAGGGTTATAAAAAAATTGTTTATAAACTTATACCTGGCGCAAATAAAATGTTACCTCGTGTTTTCTTTTCTGCTAAATCTATAAAATTTTATAATCCTAGTGAAGATATACTTATAATAAGAAATCATTCCACACATACAAAAAATGGTAGTCCTCAAAAAGGATATGAAAAATTTGAGTTTAATATTGAAGATTGCCGAAAATTTATAGATTTTTGTAAACAGTCTATAAGTAAGCATCCGGAGTGGAAAGATTTTGGATTTAGATTTTCTGATACTCAAAGATATAATTCTATAGGTGAATTTTATAGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATATCAGAGAGCTATATTGATAGCGTAGTTAATCAGGGTAAATTGTACCTATTCCAAATCTATAATAAAGATTTTTCAGCTTATAGCAAAGGGCGACCAAATCTACATACTTTATATTGGAAAGCGCTGTTTGATGAGAGAAATCTTCAAGATGTGGTTTATAAGCTAAATGGTGAGGCAGAGCTTTTTTATCGTAAACAATCAATACCTAAAAAAATCACTCACCCAGCTAAAGAGGCAATAGCTAATAAAAACAAAGATAATCCTAAAAAAGAGAGTGTTTTTGAATATGATTTAATCAAAGATAAACGCTTTACTGAAGATAAGTTTTTCTTTCACTGTCCTATTACAATCAATTTTAAATCTAGTGGAGCTAATAAGTTTAATGATGAAATCAATTTATTGCTAAAAGAAAAAGCAAATGATGTTCATATATTAAGTATAGCAAGAGGTGAAAGACATTTAGCTTACTATACTTTGGTAGATGGTAAAGGCAATATCATCAAACAAGATACTTTCAACATCATTGGTAATGATAGAATGAAAACAAACTACCATGATAAGCTTGCTGCAATAGAGAAAGATAGGGATTCAGCTAGGAAAGACTGGAAAAAGATAAATAACATCAAAGAGATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAAATAGCTAAGCTAGTTATAGAGTATAATGCTATTGTGGTTTTTGAGGATTTAAATTTTGGATTTAAAAGAGGGCGTTTCAAGGTAGAGAAGCAGGTCTATCAAAAGTTAGAAAAAATGCTAATTGAGAAACTAAACTATCTAGTTTTCAAAGATAATGAGTTTGATAAAACTGGGGGAGTGCTTAGAGCTTATCAGCTAACAGCACCTTTTGAGACTTTTAAAAAGATGGGTAAACAAACAGGTATTATCTACTATGTACCAGCTGGTTTTACTTCAAAAATTTGTCCTGTAACTGGTTTTGTAAATCAGTTATATCCTAAGTATGAAAGTGTCAGCAAATCTCAAGAGTTCTTTAGTAAGTTTGACAAGATTTGTTATAACCTTGATAAGGGCTATTTTGAGTTTAGTTTTGATTATAAAAACTTTGGTGACAAGGCTGCCAAAGGCAAGTGGACTATAGCTAGCTTTGGGAGTAGATTGATTAACTTTAGAAATTCAGATAAAAATCATAATTGGGATACTCGAGAAGTTTATCCAACTAAAGAGTTGGAGAAATTGCTAAAAGATTATTCTATCGAATATGGGCATGGCGAATGTATCAAAGCAGCTATTTGCGGTGAGAGCGACAAAAAGTTTTTTGCTAAGCTAACTAGTGTCCTAAATACTATCTTACAAATGCGTAACTCAAAAACAGGTACTGAGTTAGATTATCTAATTTCACCAGTAGCAGATGTAAATGGCAATTTCTTTGATTCGCGACAGGCGCCAAAAAATATGCCTCAAGATGCTGATGCCAATGGTGCTTATCATATTGGGCTAAAAGGTCTGATGCTACTAGGTAGGATCAAAAATAATCAAGAGGGCAAAAAACTCAATTTGGTTATCAAAAATGAAGAGTATTTTGAGTTCGTGCAGAATAGGAATAAC。
TABLE 2 PAM preference analysis
(continuation table 2)
EXAMPLE III
The base editing efficiency of Escherichia coli was measured by replacing the dFncpf1 gene in the above test screening system with either apobec1-dFncpf1-ugi gene (expression base editor dFncpf1-BE) or apobec 1-dbstfncpf 1-ugi gene (expression base editor dbsnfcpf 1-BE). ugi and apobec1 genes were synthesized by Genscript. After IPTG induction for 48h, collecting flora to extract plasmids, designing primers and establishing a library for second-generation sequencing.
The statistical second-generation sequencing results are shown in fig. 4, wherein it can BE seen that the base editor dbsFnCpf1-BE designed based on the mutant dbsFnCpf1 has a very high base substitution rate of C-T under 15 PAM sequences comprising PAM TTTC, and has higher editing efficiency and broader-spectrum PAM selectivity compared with the wild-type dFnCpf1-BE base editor, which means that the mutant dbsFnCpf1 can target more DNA sequences more freely.
Sequence listing
<110> institute of microbiology of Chinese academy of sciences
<120> FnCpf1 mutant for identifying PAM sequence in broad spectrum and application thereof
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 3903
<212> DNA
<213> Francisella novicida
<400> 1
atgtcaattt atcaagaatt tgttaataaa tatagtttaa gtaaaactct aagatttgag 60
ttaatcccac agggtaaaac acttgaaaac ataaaagcaa gaggtttgat tttagatgat 120
gagaaaagag ctaaagacta caaaaaggct aaacaaataa ttgataaata tcatcagttt 180
tttatagagg agatattaag ttcggtttgt attagcgaag atttattaca aaactattct 240
gatgtttatt ttaaacttaa aaagagtgat gatgataatc tacaaaaaga ttttaaaagt 300
gcaaaagata cgataaagaa acaaatatct gaatatataa aggactcaga gaaatttaag 360
aatttgttta atcaaaacct tatcgatgct aaaaaagggc aagagtcaga tttaattcta 420
tggctaaagc aatctaagga taatggtata gaactattta aagccaatag tgatatcaca 480
gatatagatg aggcgttaga aataatcaaa tcttttaaag gttggacaac ttattttaag 540
ggttttcatg aaaatagaaa aaatgtttat agtagcaatg atattcctac atctattatt 600
tataggatag tagatgataa tttgcctaaa tttctagaaa ataaagctaa gtatgagagt 660
ttaaaagaca aagctccaga agctataaac tatgaacaaa ttaaaaaaga tttggcagaa 720
gagctaacct ttgatattga ctacaaaaca tctgaagtta atcaaagagt tttttcactt 780
gatgaagttt ttgagatagc aaactttaat aattatctaa atcaaagtgg tattactaaa 840
tttaatacta ttattggtgg taaatttgta aatggtgaaa atacaaagag aaaaggtata 900
aatgaatata taaatctata ctcacagcaa ataaatgata aaacactcaa aaaatataaa 960
atgagtgttt tatttaagca aattttaagt gatacagaat ctaaatcttt tgtaattgat 1020
aagttagaag atgatagtga tgtagttaca acgatgcaaa gtttttatga gcaaatagca 1080
gcttttaaaa cagtagaaga aaaatctatt aaagaaacac tatctttatt atttgatgat 1140
ttaaaagctc aaaaacttga tttgagtaaa atttatttta aaaatgataa atctcttact 1200
gatctatcac aacaagtttt tgatgattat agtgttattg gtacagcggt actagaatat 1260
ataactcaac aaatagcacc taaaaatctt gataacccta gtaagaaaga gcaagaatta 1320
atagccaaaa aaactgaaaa agcaaaatac ttatctctag aaactataaa gcttgcctta 1380
gaagaattta ataagcatag agatatagat aaacagtgta ggtttgaaga aatacttgca 1440
aactttgcgg ctattccgat gatatttgat gaaatagctc aaaacaaaga caatttggca 1500
cagatatcta tcaaatatca aaatcaaggt aaaaaagacc tacttcaagc tagtgcggaa 1560
gatgatgtta aagctatcaa ggatctttta gatcaaacta ataatctctt acataaacta 1620
aaaatatttc atattagtca gtcagaagat aaggcaaata ttttagacaa ggatgagcat 1680
ttttatctag tatttgagga gtgctacttt gagctagcga atatagtgcc tctttataac 1740
aaaattagaa actatataac tcaaaagcca tatagtgatg agaaatttaa gctcaatttt 1800
gagaactcga ctttggctaa tggttgggat aaaaataaag agcctgacaa tacggcaatt 1860
ttatttatca aagatgataa atattatctg ggtgtgatga ataagaaaaa taacaaaata 1920
tttgatgata aagctatcaa agaaaataaa ggcgagggtt ataaaaaaat tgtttataaa 1980
cttttacctg gcgcaaataa aatgttacct aaggttttct tttctgctaa atctataaaa 2040
ttttataatc ctagtgaaga tatacttaga ataagaaatc attccacaca tacaaaaaat 2100
ggtagtcctc aaaaaggata tgaaaaattt gagtttaata ttgaagattg ccgaaaattt 2160
atagattttt ataaacagtc tataagtaag catccggagt ggaaagattt tggatttaga 2220
ttttctgata ctcaaagata taattctata gatgaatttt atagagaagt tgaaaatcaa 2280
ggctacaaac taacttttga aaatatatca gagagctata ttgatagcgt agttaatcag 2340
ggtaaattgt acctattcca aatctataat aaagattttt cagcttatag caaagggcga 2400
ccaaatctac atactttata ttggaaagcg ctgtttgatg agagaaatct tcaagatgtg 2460
gtttataagc taaatggtga ggcagagctt ttttatcgta aacaatcaat acctaaaaaa 2520
atcactcacc cagctaaaga ggcaatagct aataaaaaca aagataatcc taaaaaagag 2580
agtgtttttg aatatgattt aatcaaagat aaacgcttta ctgaagataa gtttttcttt 2640
cactgtccta ttacaatcaa ttttaaatct agtggagcta ataagtttaa tgatgaaatc 2700
aatttattgc taaaagaaaa agcaaatgat gttcatatat taagtataga cagaggtgaa 2760
agacatttag cttactatac tttggtagat ggtaaaggca atatcatcaa acaagatact 2820
ttcaacatca ttggtaatga tagaatgaaa acaaactacc atgataagct tgctgcaata 2880
gagaaagata gggattcagc taggaaagac tggaaaaaga taaataacat caaagagatg 2940
aaagagggct atctatctca ggtagttcat gaaatagcta agctagttat agagtataat 3000
gctattgtgg tttttgagga tttaaatttt ggatttaaaa gagggcgttt caaggtagag 3060
aagcaggtct atcaaaagtt agaaaaaatg ctaattgaga aactaaacta tctagttttc 3120
aaagataatg agtttgataa aactggggga gtgcttagag cttatcagct aacagcacct 3180
tttgagactt ttaaaaagat gggtaaacaa acaggtatta tctactatgt accagctggt 3240
tttacttcaa aaatttgtcc tgtaactggt tttgtaaatc agttatatcc taagtatgaa 3300
agtgtcagca aatctcaaga gttctttagt aagtttgaca agatttgtta taaccttgat 3360
aagggctatt ttgagtttag ttttgattat aaaaactttg gtgacaaggc tgccaaaggc 3420
aagtggacta tagctagctt tgggagtaga ttgattaact ttagaaattc agataaaaat 3480
cataattggg atactcgaga agtttatcca actaaagagt tggagaaatt gctaaaagat 3540
tattctatcg aatatgggca tggcgaatgt atcaaagcag ctatttgcgg tgagagcgac 3600
aaaaagtttt ttgctaagct aactagtgtc ctaaatacta tcttacaaat gcgtaactca 3660
aaaacaggta ctgagttaga ttatctaatt tcaccagtag cagatgtaaa tggcaatttc 3720
tttgattcgc gacaggcgcc aaaaaatatg cctcaagatg ctgatgccaa tggtgcttat 3780
catattgggc taaaaggtct gatgctacta ggtaggatca aaaataatca agagggcaaa 3840
aaactcaatt tggttatcaa aaatgaagag tattttgagt tcgtgcagaa taggaataac 3900
tag 3903
<210> 2
<211> 1300
<212> PRT
<213> Francisella novicida
<400> 2
MSIYQEFVNK YSLSKTLRFE LIPQGKTLEN IKARGLILDD EKRAKDYKKA KQIIDKYHQF 60
FIEEILSSVC ISEDLLQNYS DVYFKLKKSD DDNLQKDFKS AKDTIKKQIS EYIKDSEKFK 120
NLFNQNLIDA KKGQESDLIL WLKQSKDNGI ELFKANSDIT DIDEALEIIK SFKGWTTYFK 180
GFHENRKNVY SSNDIPTSII YRIVDDNLPK FLENKAKYES LKDKAPEAIN YEQIKKDLAE 240
ELTFDIDYKT SEVNQRVFSL DEVFEIANFN NYLNQSGITK FNTIIGGKFV NGENTKRKGI 300
NEYINLYSQQ INDKTLKKYK MSVLFKQILS DTESKSFVID KLEDDSDVVT TMQSFYEQIA 360
AFKTVEEKSI KETLSLLFDD LKAQKLDLSK IYFKNDKSLT DLSQQVFDDY SVIGTAVLEY 420
ITQQIAPKNL DNPSKKEQEL IAKKTEKAKY LSLETIKLAL EEFNKHRDID KQCRFEEILA 480
NFAAIPMIFD EIAQNKDNLA QISIKYQNQG KKDLLQASAE DDVKAIKDLL DQTNNLLHKL 540
KIFHISQSED KANILDKDEH FYLVFEECYF ELANIVPLYN KIRNYITQKP YSDEKFKLNF 600
ENSTLANGWD KNKEPDNTAI LFIKDDKYYL GVMNKKNNKI FDDKAIKENK GEGYKKIVYK 660
LLPGANKMLP KVFFSAKSIK FYNPSEDILR IRNHSTHTKN GSPQKGYEKF EFNIEDCRKF 720
IDFYKQSISK HPEWKDFGFR FSDTQRYNSI DEFYREVENQ GYKLTFENIS ESYIDSVVNQ 780
GKLYLFQIYN KDFSAYSKGR PNLHTLYWKA LFDERNLQDV VYKLNGEAEL FYRKQSIPKK 840
ITHPAKEAIA NKNKDNPKKE SVFEYDLIKD KRFTEDKFFF HCPITINFKS SGANKFNDEI 900
NLLLKEKAND VHILSIDRGE RHLAYYTLVD GKGNIIKQDT FNIIGNDRMK TNYHDKLAAI 960
EKDRDSARKD WKKINNIKEM KEGYLSQVVH EIAKLVIEYN AIVVFEDLNF GFKRGRFKVE 1020
KQVYQKLEKM LIEKLNYLVF KDNEFDKTGG VLRAYQLTAP FETFKKMGKQ TGIIYYVPAG 1080
FTSKICPVTG FVNQLYPKYE SVSKSQEFFS KFDKICYNLD KGYFEFSFDY KNFGDKAAKG 1140
KWTIASFGSR LINFRNSDKN HNWDTREVYP TKELEKLLKD YSIEYGHGEC IKAAICGESD 1200
KKFFAKLTSV LNTILQMRNS KTGTELDYLI SPVADVNGNF FDSRQAPKNM PQDADANGAY 1260
HIGLKGLMLL GRIKNNQEGK KLNLVIKNEE YFEFVQNRNN 1300
<210> 3
<211> 750
<212> DNA
<213> Francisella novicida
<400> 3
ggtaaaaaag acctacttca agctagtgcg gaagatgatg ttaaagctat caaggatctt 60
ttagatcaaa ctaataatct cttacataaa ctaaaaatat ttcatattag tcagtcagaa 120
gataaggcaa atattttaga caaggatgag catttttatc tagtatttga ggagtgctac 180
tttgagctag cgaatatagt gcctctttat aacaaaatta gaaactatat aactcaaaag 240
ccatatagtg atgagaaatt taagctcaat tttgagaact cgactttggc taatggttgg 300
gataaaaata aagagcctga caatacggca attttattta tcaaagatga taaatattat 360
ctgggtgtga tgaataagaa aaataacaaa atatttgatg ataaagctat caaagaaaat 420
aaaggcgagg gttataaaaa aattgtttat aaacttttac ctggcgcaaa taaaatgtta 480
cctaaggttt tcttttctgc taaatctata aaattttata atcctagtga agatatactt 540
agaataagaa atcattccac acatacaaaa aatggtagtc ctcaaaaagg atatgaaaaa 600
tttgagttta atattgaaga ttgccgaaaa tttatagatt tttataaaca gtctataagt 660
aagcatccgg agtggaaaga ttttggattt agattttctg atactcaaag atataattct 720
atagatgaat tttatagaga agttgaaaat 750
<210> 4
<211> 3903
<212> DNA
<213> Francisella novicida
<400> 4
atgtcaattt atcaagaatt tgttaataaa tatagtttaa gtaaaactct aagatttgag 60
ttaatcccac agggtaaaac acttgaaaac ataaaagcaa gaggtttgat tttagatgat 120
gagaaaagag ctaaagacta caaaaaggct aaacaaataa ttgataaata tcatcagttt 180
tttatagagg agatattaag ttcggtttgt attagcgaag atttattaca aaactattct 240
gatgtttatt ttaaacttaa aaagagtgat gatgataatc tacaaaaaga ttttaaaagt 300
gcaaaagata cgataaagaa acaaatatct gaatatataa aggactcaga gaaatttaag 360
aatttgttta atcaaaacct tatcgatgct aaaaaagggc aagagtcaga tttaattcta 420
tggctaaagc aatctaagga taatggtata gaactattta aagccaatag tgatatcaca 480
gatatagatg aggcgttaga aataatcaaa tcttttaaag gttggacaac ttattttaag 540
ggttttcatg aaaatagaaa aaatgtttat agtagcaatg atattcctac atctattatt 600
tataggatag tagatgataa tttgcctaaa tttctagaaa ataaagctaa gtatgagagt 660
ttaaaagaca aagctccaga agctataaac tatgaacaaa ttaaaaaaga tttggcagaa 720
gagctaacct ttgatattga ctacaaaaca tctgaagtta atcaaagagt tttttcactt 780
gatgaagttt ttgagatagc aaactttaat aattatctaa atcaaagtgg tattactaaa 840
tttaatacta ttattggtgg taaatttgta aatggtgaaa atacaaagag aaaaggtata 900
aatgaatata taaatctata ctcacagcaa ataaatgata aaacactcaa aaaatataaa 960
atgagtgttt tatttaagca aattttaagt gatacagaat ctaaatcttt tgtaattgat 1020
aagttagaag atgatagtga tgtagttaca acgatgcaaa gtttttatga gcaaatagca 1080
gcttttaaaa cagtagaaga aaaatctatt aaagaaacac tatctttatt atttgatgat 1140
ttaaaagctc aaaaacttga tttgagtaaa atttatttta aaaatgataa atctcttact 1200
gatctatcac aacaagtttt tgatgattat agtgttattg gtacagcggt actagaatat 1260
ataactcaac aaatagcacc taaaaatctt gataacccta gtaagaaaga gcaagaatta 1320
atagccaaaa aaactgaaaa agcaaaatac ttatctctag aaactataaa gcttgcctta 1380
gaagaattta ataagcatag agatatagat aaacagtgta ggtttgaaga aatacttgca 1440
aactttgcgg ctattccgat gatatttgat gaaatagctc aaaacaaaga caatttggca 1500
cagatatcta tcaaatatca aaatcaaggt aaaaaagacc tacttcaagc tagtgcggaa 1560
gatgatgtta aagctatcaa ggatctttta gatcaaacta ataatctctt acataaacta 1620
aaaatatttc atattagtca gtcagaagat aaggcaaata ttttagacaa ggatgagcat 1680
ttttatctag tatttgtgga gtgctacctt gagctagcga atatagtgcc tctttataac 1740
aaaattagaa actatataac tcaaaagcca tatagtgatg agaaatttaa gctcaatttt 1800
gagaactcga ctttggctaa tggttgggat aaaaataatg agcctgacaa tacggcaatt 1860
ttatttatca aagatgataa atattatctg ggtgtgatga ataagaaaaa taacaaaata 1920
tttgatgata aagctatcaa agaaaataaa ggcgagggtt ataaaaaaat tgtttataaa 1980
cttatacctg gcgcaaataa aatgttacct cgtgttttct tttctgctaa atctataaaa 2040
ttttataatc ctagtgaaga tatacttata ataagaaatc attccacaca tacaaaaaat 2100
ggtagtcctc aaaaaggata tgaaaaattt gagtttaata ttgaagattg ccgaaaattt 2160
atagattttt gtaaacagtc tataagtaag catccggagt ggaaagattt tggatttaga 2220
ttttctgata ctcaaagata taattctata ggtgaatttt atagagaagt tgaaaatcaa 2280
ggctacaaac taacttttga aaatatatca gagagctata ttgatagcgt agttaatcag 2340
ggtaaattgt acctattcca aatctataat aaagattttt cagcttatag caaagggcga 2400
ccaaatctac atactttata ttggaaagcg ctgtttgatg agagaaatct tcaagatgtg 2460
gtttataagc taaatggtga ggcagagctt ttttatcgta aacaatcaat acctaaaaaa 2520
atcactcacc cagctaaaga ggcaatagct aataaaaaca aagataatcc taaaaaagag 2580
agtgtttttg aatatgattt aatcaaagat aaacgcttta ctgaagataa gtttttcttt 2640
cactgtccta ttacaatcaa ttttaaatct agtggagcta ataagtttaa tgatgaaatc 2700
aatttattgc taaaagaaaa agcaaatgat gttcatatat taagtatagc aagaggtgaa 2760
agacatttag cttactatac tttggtagat ggtaaaggca atatcatcaa acaagatact 2820
ttcaacatca ttggtaatga tagaatgaaa acaaactacc atgataagct tgctgcaata 2880
gagaaagata gggattcagc taggaaagac tggaaaaaga taaataacat caaagagatg 2940
aaagagggct atctatctca ggtagttcat gaaatagcta agctagttat agagtataat 3000
gctattgtgg tttttgagga tttaaatttt ggatttaaaa gagggcgttt caaggtagag 3060
aagcaggtct atcaaaagtt agaaaaaatg ctaattgaga aactaaacta tctagttttc 3120
aaagataatg agtttgataa aactggggga gtgcttagag cttatcagct aacagcacct 3180
tttgagactt ttaaaaagat gggtaaacaa acaggtatta tctactatgt accagctggt 3240
tttacttcaa aaatttgtcc tgtaactggt tttgtaaatc agttatatcc taagtatgaa 3300
agtgtcagca aatctcaaga gttctttagt aagtttgaca agatttgtta taaccttgat 3360
aagggctatt ttgagtttag ttttgattat aaaaactttg gtgacaaggc tgccaaaggc 3420
aagtggacta tagctagctt tgggagtaga ttgattaact ttagaaattc agataaaaat 3480
cataattggg atactcgaga agtttatcca actaaagagt tggagaaatt gctaaaagat 3540
tattctatcg aatatgggca tggcgaatgt atcaaagcag ctatttgcgg tgagagcgac 3600
aaaaagtttt ttgctaagct aactagtgtc ctaaatacta tcttacaaat gcgtaactca 3660
aaaacaggta ctgagttaga ttatctaatt tcaccagtag cagatgtaaa tggcaatttc 3720
tttgattcgc gacaggcgcc aaaaaatatg cctcaagatg ctgatgccaa tggtgcttat 3780
catattgggc taaaaggtct gatgctacta ggtaggatca aaaataatca agagggcaaa 3840
aaactcaatt tggttatcaa aaatgaagag tattttgagt tcgtgcagaa taggaataac 3900
tag 3903
Claims (10)
1. A mutant of CRISPR nuclease FnCpf1, which has the following mutations relative to wild-type FnCpf1 of the amino acid sequence shown as SEQ ID NO: 2:
K671R/E566V/D751G/N508H/N637S、
K671R/E566V/D751G/F570L/N634D/R755K、
K671R/E566V/D751G/S518G/K639R、
K671R/E566V/D751G/F570L/E686D、
K671R/E566V/D751G/K613N/N637S/N534K/G664V、
K671R/E566V/D751G/K613N/F570L/G664S/N637Y、
K671R/E566V/D751G/K613N/Y724C/F570L, or
K671R/E566V/D751G/K613N/Y724C/F570L/R690I/L662I。
2. The mutant of CRISPR nuclease FnCpf1 of claim 1, wherein the following mutations are present relative to wild-type dFnCpf 1:
K671R/E566V/D751G/K613N/Y724C/F570L/R690I/L662I、
K671R/E566V/D751G/K613N/N637S/N534K/G664V、
K671R/E566V/D751G/K613N/F570L/G664S/N637Y、
K671R/E566V/D751G/K613N/Y724C/F570L。
3. the mutant of CRISPR nuclease FnCpf1 of claim 2, wherein the following mutations are present relative to wild-type FnCpf 1:
K671R/E566V/D751G/K613N/Y724C/F570L/R690I/L662I。
4. a gene encoding the mutant as claimed in any one of claims 1 to 3.
5. The encoding gene of claim 4, wherein the nucleotide sequence is as shown in SEQ ID NO. 4.
6. A vector comprising the gene encoding the gene according to claim 4 or 5.
7. The vector of claim 6, which is a vector for gene editing.
8. A recombinant cell line comprising a gene encoding the gene of claim 4 or 5.
9. Use of the coding gene of claim 4 or 5 for gene editing.
10. Use according to claim 9 for the genetic editing of the bacterial genome.
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| CN113005141A (en) * | 2021-01-05 | 2021-06-22 | 温州医科大学 | Gene editing tool composed of high-activity mutant, preparation method and method for repairing congenital retinoschisis disease pathogenic gene |
| WO2022061748A1 (en) * | 2020-09-25 | 2022-03-31 | 中国科学院微生物研究所 | Fncpf1 mutant for broad-spectrum identification on pam sequence and use thereof |
| WO2024060814A1 (en) * | 2022-09-21 | 2024-03-28 | 香港中文大学（深圳） | Cas12a variant and use thereof in gene editing |
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