Patent Publication Number: US-2023146044-A1

Title: Fncpf1 mutant for broad-spectrum identification on pam sequence ans use thereof

Description:
TECHNICAL FIELD 
     The invention relates to the field of biotechnology. In particular, the invention relates to mutants of CRISPR nuclease FnCpfI and its application. 
     BACKGROUND OF THE INVENTION 
     CRISPR-Cas system has been a powerful genome editing tool to insert, delete, or modify the sequences of genome in a living organism since its conception. In order to improve the accuracy and editing efficiency of the fixed-point mutation, a single-base editing system combining CRISPR-Cas proteins with cytosine/adenine deaminase is recently developed as a new generation of more accurate gene-editing tools. The single-base editing technology can accurately and irreversibly transform from one base pair to another (C-to-T or A-to-G) without causing DNA double-strand breaks and homologous recombination. However, the limited availability of editable sites in the target bacterial genome and off-target activity have restrained the CRISPR/Cas BE system&#39;s applicability. 
     As an alternative CRISPR nuclease beyond the commonly used Cas9, Cpf1 offers potentially advantageous in multiplex gene targeting in the same cell due to its RNA endonuclease activity. The ability to simultaneously manipulate multiple genes is highly demanded to the system level, which enables the interrogation of much more complex interactions in genome-scale networks. Besides, the Cpf1 system displays some enticing features such as a more concise crRNA (˜40 nt), smaller molecular weight, as well as low rates of off-target activity. Therefore, we believe that DNA enzyme inactivation of Cpf1 (dCpf1) has great potential as an effective tool for multi-gene regulation. 
     Nevertheless, Cpf1 mediated gene editing essentially requires the recognition of a T-rich PAM of form 5′-TTTV/TTV (V represents A, C, or G), which hinders its application in gene editing of GC-rich organisms. To address this limitation, two  Acidaminococcus  sp. Cpf1 (AsCpf1) variants RVR and RR were initially engineered to recognize alternative PAMs, i.e., TATV and TYCV, respectively. (Gao L, Cox D B T, Yan W X, Manteiga J C, Schneider M W, Yamano T, et al. Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol 2017; 35:789-92.) Later, the target range of AsCpf1 was further expanded to TTYN/VTTV/TRTV PAMs. (Kleinstiver B P, Sousa A A, Walton R T, Tak Y E, Hsu J Y, Clement K, et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol 2019; 37: 276-82.) Besides, another widely used Cpf1 from Francisellanovicida (FnCpf1), was engineered to identify non-canonical PAMs, however the −4 T preference in the PAM sequence was ignored in the study. (Wang L, Wang H, Liu H, Zhao Q, Liu B, Wang L, et al. Improved CRISPR-Cas12a-assisted one-pot DNA editing method enables seamless DNA editing. Biotechnol Bioeng 2019; 116: 1463-74.) Nevertheless, many PAMs remain inaccessible to Cpf1, especially the GC-rich PAM sequences. Thus, additional variants with expanded targeting capabilities are needed to enable applications requiring high targeting density and flexibility. 
     SUMMARY OF THE INVENTION 
     In view of the shortcomings of the existing technology, we designed a negative screening assay in  E. coli  to extend the PAM sequence preference for dFnCpf1 to the GC-rich PAMs. The resulting subset of dFnCpf1 mutants exhibited a higher recognition and binding for sites with non-canonical PAMs and retained robust activities on canonical TTTV PAMs. 
     The present invention provides a mutant of CRISPR nuclease FnCpf1, which exists the following mutations relative to wild-type FnCpf1 with amino acid sequence as shown in 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, K671R/E566V/D751G/K613N/Y724C/F570L/R690I/L662I. 
     Preferably, the mutant has the following mutations relative to wild-type FnCpf1: 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 
     Optimally, the mutation has the following mutations relative to wild-type FnCpf1: K671R/E566V/D751G/K613N/Y724C/F570L/R690I/L662I 
     The invention further provides the coding gene of the mutants mentioned above. The nucleotide sequence is shown in SEQ ID NO:4. 
     The invention also provides vectors containing the above genes for gene editing. 
     Further, we provide a recombinant cell line containing the vectors, such as  Escherichia coli , is provided. 
     The invention also provides applications of the aforementioned genes in gene editing such as base editing of bacterial genomes. 
     The mutants in our invention exhibit higher recognition and binding ability at sites of non-canonical PAMs compared with wild-type FnCpf1, while still maintaining robust activity at typical TTTV PAMs. Furthermore, we demonstrated that the mutants in our invention could be designed as cytosine base editor in multi-target genome editing in  E. coli  with higher efficiency and broader targeting range than wild-type FnCpf1. It indicates that the mutant in our invention has obvious advantages in the recognition range of PAM. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1   . Schematic representation of a dCpf1-interference YFP-based repression circuit in  E. coli . In the circuit, dCpf1 was induced by IPTG and crRNA were expressed from a constitutive promoter (J23119), and a reporter gene YFP is repressed by the dFnCpf1-crRNA complex in the upstream region of its initial transcription. Therefore, the ability of dCpf1 mutants to recognize and bind PAM sequences can be quantified by the inhibition of YFP fluorescence values. 
         FIG.  2   . Flow chart of the directed evolution of CRISPR nuclease dFnCpf1. The directed evolution process was to use error-prone PCR to construct dFnCpf1 mutation library, use flow cytometry to screen the bacteria with significantly reduced YFP fluorescence value and to further verify its inhibition efficiency. Then sequencing, and obtain the best mutant for the next round of directed evolution. 
         FIG.  3   . PAM preference profiles for WT dFnCpf1 and eight selected mutants. The variants with the highest activity in the corresponding PAM trajectories were assessed for the global 64 PAM preferences (NNNV, V is not included due to the weak preference) and later compared with WT dFnCpf1. The mutants 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 recognize PAM GCCG, CCGC, GCGC and CGCC (SCSC), respectively. The mutants dFnCpf1 (VRGN/N637 S/N534K/G664V), dFnCpf1 (VRGN/F570L/G664S/N637Y), dFnCpf1 (VRGN/Y724C/F570L), dFnCpf1 (VRGN/Y724C/F570L/R690I/L662I) were selected from mutants obtained by directed evolution that recognize PAM CGCC, GGCC, CGGC and GGGC (SGSC), respectively. VRG refers to the mutations E566V, K671R and D751G, besides N refers to the additional mutation K613N. YFP fluorescence intensity after 200 μM IPTG induction was used as the characterization value of PAM preference profiles. 
         FIG.  4   . Base editing mediated by bsdFnCpf1-BE and dFnCpf1-BE in  E. coli . Determination of bsdFnCpf1-BE or dFnCpf1-BE induced base-editing frequency at the target site “GGGCACTCTCCAGATAGGGAT” with different PAMs. Comparison of C to T editing efficiency of C8 at the target site between bsdFnCpf1-BE and dFnCpf1-BE, assessed by targeted deep sequencing. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In the following, the invention is further elaborated through the implementation scheme of research process to better understand the invention, but does not constitute a limitation on the invention. 
     We selected dFnCpf1 from Francisellanovicidafor directed evolution, and focused on the −2 to −4 bits of the PAM sequence (the −1 bit base was not considered because of the large selectivity), then tried to extend the PAM sequence preference to the GC-rich PAMs. 
     Sequences of dFnCpf1 is provided as follows (SEQ ID NO:1): 
     
       
         
           
               
               
            
               
                 ATGTCAATTTATCAAGAATTTGTTAATAAATATAGTTTAAGTAAAACTCTAAGATTTG 
                   
               
               
                   
               
               
                 AGTTAATCCCACAGGGTAAAACACTTGAAAACATAAAAGCAAGAGGTTTGATTTT 
               
               
                   
               
               
                 AGATGATGAGAAAAGAGCTAAAGACTACAAAAAGGCTAAACAAATAATTGATAAA 
               
               
                   
               
               
                 TATCATCAGTTTTTTATAGAGGAGATATTAAGTTCGGTTTGTATTAGCGAAGATTTAT 
               
               
                   
               
               
                 TACAAAACTATTCTGATGTTTATTTTAAACTTAAAAAGAGTGATGATGATAATCTAC 
               
               
                   
               
               
                 AAAAAGATTTTAAAAGTGCAAAAGATACGATAAAGAAACAAATATCTGAATATATA 
               
               
                   
               
               
                 AAGGACTCAGAGAAATTTAAGAATTTGTTTAATCAAAACCTTATCGATGCTAAAAA 
               
               
                   
               
               
                 AGGGCAAGAGTCAGATTTAATTCTATGGCTAAAGCAATCTAAGGATAATGGTATAG 
               
               
                   
               
               
                 AACTATTTAAAGCCAATAGTGATATCACAGATATAGATGAGGCGTTAGAAATAATCA 
               
               
                   
               
               
                 AATCTTTTAAAGGTTGGACAACTTATTTTAAGGGTTTTCATGAAAATAGAAAAAAT 
               
               
                   
               
               
                 GTTTATAGTAGCAATGATATTCCTACATCTATTATTTATAGGATAGTAGATGATAATTT 
               
               
                   
               
               
                 GCCTAAATTTCTAGAAAATAAAGCTAAGTATGAGAGTTTAAAAGACAAAGCTCCA 
               
               
                   
               
               
                 GAAGCTATAAACTATGAACAAATTAAAAAAGATTTGGCAGAAGAGCTAACCTTTG 
               
               
                   
               
               
                 ATATTGACTACAAAACATCTGAAGTTAATCAAAGAGTTTTTTCACTTGATGAAGTT 
               
               
                   
               
               
                 TTTGAGATAGCAAACTTTAATAATTATCTAAATCAAAGTGGTATTACTAAATTTAATA 
               
               
                   
               
               
                 CTATTATTGGTGGTAAATTTGTAAATGGTGAAAATACAAAGAGAAAAGGTATAAAT 
               
               
                   
               
               
                 GAATATATAAATCTATACTCACAGCAAATAAATGATAAAACACTCAAAAAATATAAA 
               
               
                   
               
               
                 ATGAGTGTTTTATTTAAGCAAATTTTAAGTGATACAGAATCTAAATCTTTTGTAATT 
               
               
                   
               
               
                 GATAAGTTAGAAGATGATAGTGATGTAGTTACAACGATGCAAAGTTTTTATGAGCA 
               
               
                   
               
               
                 AATAGCAGCTTTTAAAACAGTAGAAGAAAAATCTATTAAAGAAACACTATCTTTAT 
               
               
                   
               
               
                 TATTTGATGATTTAAAAGCTCAAAAACTTGATTTGAGTAAAATTTATTTTAAAAATG 
               
               
                   
               
               
                 ATAAATCTCTTACTGATCTATCACAACAAGTTTTTGATGATTATAGTGTTATTGGTAC 
               
               
                   
               
               
                 AGCGGTACTAGAATATATAACTCAACAAATAGCACCTAAAAATCTTGATAACCCTA 
               
               
                   
               
               
                 GTAAGAAAGAGCAAGAATTAATAGCCAAAAAAACTGAAAAAGCAAAATACTTATC 
               
               
                   
               
               
                 TCTAGAAACTATAAAGCTTGCCTTAGAAGAATTTAATAAGCATAGAGATATAGATAA 
               
               
                   
               
               
                 ACAGTGTAGGTTTGAAGAAATACTTGCAAACTTTGCGGCTATTCCGATGATATTTG 
               
               
                   
               
               
                 ATGAAATAGCTCAAAACAAAGACAATTTGGCACAGATATCTATCAAATATCAAAAT 
               
               
                   
               
               
                 CAAGGTAAAAAAGACCTACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCTATCA 
               
               
                   
               
               
                 AGGATCTTTTAGATCAAACTAATAATCTCTTACATAAACTAAAAATATTTCATATTAG 
               
               
                   
               
               
                 TCAGTCAGAAGATAAGGCAAATATTTTAGACAAGGATGAGCATTTTTATCTAGTATT 
               
               
                   
               
               
                 TGAGGAGTGCTACTTTGAGCTAGCGAATATAGTGCCTCTTTATAACAAAATTAGAA 
               
               
                   
               
               
                 ACTATATAACTCAAAAGCCATATAGTGATGAGAAATTTAAGCTCAATTTTGAGAACT 
               
               
                   
               
               
                 CGACTTTGGCTAATGGTTGGGATAAAAATAAAGAGCCTGACAATACGGCAATTTTA 
               
               
                   
               
               
                 TTTATCAAAGATGATAAATATTATCTGGGTGTGATGAATAAGAAAAATAACAAAATA 
               
               
                   
               
               
                 TTTGATGATAAAGCTATCAAAGAAAATAAAGGCGAGGGTTATAAAAAAATTGTTTA 
               
               
                   
               
               
                 TAAACTTTTACCTGGCGCAAATAAAATGTTACCTAAGGTTTTCTTTTCTGCTAAATC 
               
               
                   
               
               
                 TATAAAATTTTATAATCCTAGTGAAGATATACTTAGAATAAGAAATCATTCCACACAT 
               
               
                   
               
               
                 ACAAAAAATGGTAGTCCTCAAAAAGGATATGAAAAATTTGAGTTTAATATTGAAGA 
               
               
                   
               
               
                 TTGCCGAAAATTTATAGATTTTTATAAACAGTCTATAAGTAAGCATCCGGAGTGGAA 
               
               
                   
               
               
                 AGATTTTGGATTTAGATTTTCTGATACTCAAAGATATAATTCTATAGATGAATTTTAT 
               
               
                   
               
               
                 AGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATATCAGAGAGCT 
               
               
                   
               
               
                 ATATTGATAGCGTAGTTAATCAGGGTAAATTGTACCTATTCCAAATCTATAATAAAGA 
               
               
                   
               
               
                 TTTTTCAGCTTATAGCAAAGGGCGACCAAATCTACATACTTTATATTGGAAAGCGCT 
               
               
                   
               
               
                 GTTTGATGAGAGAAATCTTCAAGATGTGGTTTATAAGCTAAATGGTGAGGCAGAGC 
               
               
                   
               
               
                 TTTTTTATCGTAAACAATCAATACCTAAAAAAATCACTCACCCAGCTAAAGAGGCA 
               
               
                   
               
               
                 ATAGCTAATAAAAACAAAGATAATCCTAAAAAAGAGAGTGTTTTTGAATATGATTT 
               
               
                   
               
               
                 AATCAAAGATAAACGCTTTACTGAAGATAAGTTTTTCTTTCACTGTCCTATTACAAT 
               
               
                   
               
               
                 CAATTTTAAATCTAGTGGAGCTAATAAGTTTAATGATGAAATCAATTTATTGCTAAA 
               
               
                   
               
               
                 AGAAAAAGCAAATGATGTTCATATATTAAGTATAGACAGAGGTGAAAGACATTTAG 
               
               
                   
               
               
                 CTTACTATACTTTGGTAGATGGTAAAGGCAATATCATCAAACAAGATACTTTCAACA 
               
               
                   
               
               
                 TCATTGGTAATGATAGAATGAAAACAAACTACCATGATAAGCTTGCTGCAATAGAG 
               
               
                   
               
               
                 AAAGATAGGGATTCAGCTAGGAAAGACTGGAAAAAGATAAATAACATCAAAGAG 
               
               
                   
               
               
                 ATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAAATAGCTAAGCTAGTTATAGA 
               
               
                   
               
               
                 GTATAATGCTATTGTGGTTTTTGAGGATTTAAATTTTGGATTTAAAAGAGGGCGTTT 
               
               
                   
               
               
                 CAAGGTAGAGAAGCAGGTCTATCAAAAGTTAGAAAAAATGCTAATTGAGAAACTA 
               
               
                   
               
               
                 AACTATCTAGTTTTCAAAGATAATGAGTTTGATAAAACTGGGGGAGTGCTTAGAGC 
               
               
                   
               
               
                 TTATCAGCTAACAGCACCTTTTGAGACTTTTAAAAAGATGGGTAAACAAACAGGT 
               
               
                   
               
               
                 ATTATCTACTATGTACCAGCGGTTTTACTTCAAAAATTTGTCCTGTAACTGGTTTTG 
               
               
                   
               
               
                 TAAATCAGTTATATCCTAAGTATGAAAGTGTCAGCAAATCTCAAGAGTTCTTTAGTA 
               
               
                   
               
               
                 AGTTTGACAAGATTTGTTATAACCTTGATAAGGGCTATTTTGAGTTTAGTTTTGATT 
               
               
                   
               
               
                 ATAAAAACTTTGGTGACAAGGCTGCCAAAGGCAAGTGGACTATAGCTAGCTTTGG 
               
               
                   
               
               
                 GAGTAGATTGATTAACTTTAGAAATTCAGATAAAAATCATAATTGGGATACTCGAG 
               
               
                   
               
               
                 AAGTTTATCCAACTAAAGAGTTGGAGAAATTGCTAAAAGATTATTCTATCGAATATG 
               
               
                   
               
               
                 GGCATGGCGAATGTATCAAAGCAGCTATTTGCGGTGAGAGCGACAAAAAGTTTTT 
               
               
                   
               
               
                 TGCTAAGCTAACTAGTGTCCTAAATACTATCTTACAAATGCGTAACTCAAAAACAG 
               
               
                   
               
               
                 GTACTGAGTTAGATTATCTAATTTCACCAGTAGCAGATGTAAATGGCAATTTCTTTG 
               
               
                   
               
               
                 ATTCGCGACAGGCGCCAAAAAATATGCCTCAAGATGCTGATGCCAATGGTGCTTAT 
               
               
                   
               
               
                 CATATTGGGCTAAAAGGTCTGATGCTACTAGGTAGGATCAAAAATAATCAAGAGGG 
               
               
                   
               
               
                 CAAAAAACTCAATTTGGTTATCAAAAATGAAGAGTATTTTGAGTTCGTGCAGAATA 
               
               
                   
               
               
                 GGAATAACTAG. 
               
            
           
         
       
     
     A 750 bp DNA sequence which is randomly mutagenized through error-prone PCR is provided as follow (SEQ ID NO: 3): 
     
       
         
           
               
            
               
                 GGTAAAAAAGACCTACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCTAT 
               
               
                   
               
               
                 CAAGGATCTTTTAGATCAAACTAATAATCTCTTACATAAACTAAAAATAT 
               
               
                   
               
               
                 TTCATATTAGTCAGTCAGAAGATAAGGCAAATATTTTAGACAAGGATGAG 
               
               
                   
               
               
                 CATTTTTATCTAGTATTTGAGGAGTGCTACTTTGAGCTAGCGAATATAGT 
               
               
                   
               
               
                 GCCTCTTTATAACAAAATTAGAAACTATATAACTCAAAAGCCATATAGTG 
               
               
                   
               
               
                 ATGAGAAATTTAAGCTCAATTTTGAGAACTCGACTTTGGCTAATGGTTGG 
               
               
                   
               
               
                 GATAAAAATAAAGAGCCTGACAATACGGCAATTTTATTTATCAAAGATGA 
               
               
                   
               
               
                 TAAATATTATCTGGGTGTGATGAATAAGAAAAATAACAAAATATTTGATG 
               
               
                   
               
               
                 ATAAAGCTATCAAAGAAAATAAAGGCGAGGGTTATAAAAAAATTGTTTAT 
               
               
                   
               
               
                 AAACTTTTACCTGGCGCAAATAAAATGTTACCTAAGGTTTTCTTTTCTGC 
               
               
                   
               
               
                 TAAATCTATAAAATTTTATAATCCTAGTGAAGATATACTTAGAATAAGAA 
               
               
                   
               
               
                 ATCATTCCACACATACAAAAAATGGTAGTCCTCAAAAAGGATATGAAAAA 
               
               
                   
               
               
                 TTTGAGTTTAATATTGAAGATTGCCGAAAATTTATAGATTTTTATAAACA 
               
               
                   
               
               
                 GTCTATAAGTAAGCATCCGGAGTGGAAAGATTTTGGATTTAGATTTTCTG 
               
               
                   
               
               
                 ATACTCAAAGATATAATTCTATAGATGAATTTTATAGAGAAGTTGAAAAT. 
               
            
           
         
       
     
     The above DNA sequence encodes 1300 amino acid residues, and the amino acid sequence (SEQ ID NO: 2) is as follows: 
     
       
         
           
               
               
            
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYH 
                   
               
               
                   
               
               
                 QFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSE 
               
               
                   
               
               
                 KFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWT 
               
               
                   
               
               
                 TYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK 
               
               
                   
               
               
                 KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGEN 
               
               
                   
               
               
                 TKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTM 
               
               
                   
               
               
                 QSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY 
               
               
                   
               
               
                 SVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDI 
               
               
                   
               
               
                 DKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIK 
               
               
                   
               
               
                 DLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYI 
               
               
                   
               
               
                 TQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFD 
               
               
                   
               
               
                 DKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVEN 
               
               
                   
               
               
                 QGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNL 
               
               
                   
               
               
                 QDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE 
               
               
                   
               
               
                 DKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGN 
               
               
                   
               
               
                 IIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHE 
               
               
                   
               
               
                 IAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKT 
               
               
                   
               
               
                 GGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQ 
               
               
                   
               
               
                 EFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNW 
               
               
                   
               
               
                 DTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSK 
               
               
                   
               
               
                 TGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQ 
               
               
                   
               
               
                 EGKKLNLVIKNEEYFEFVQNRNN. 
               
            
           
         
       
     
     Embodiment 1 
     First of all, we developed a bacterial negative screening assay where a constitutively expressed yfpgene was targeted in the upstream region of its initial transcription by a crRNA. Gibson Assembly or Golden Gate Assembly was employed to construct plasmids used in this study. The plasmid sequences were confirmed via Sanger sequencing. A 750 bp DNA sequence (SEQ ID NO: 3) from dFnCpf1 was randomly mutagenized through error-prone PCR to construct a dfncpf1 variants library. Later, the PCR products were inserted into the modified the Repressor Generator Plasmid (RGP) containing a pTac inducible promoter, a p15A replication origin, and an ampicillin-selectable marker using the Golden Gate method. The vector was used to control the inducible expression of dCpf1 enzymes. The crRNA plasmid contained a synthetic constitutive promoter J23119, a ColE1 replication origin, and a chloramphenicol-selectable marker for crRNA expression. The reporter plasmid contained a pSC101 replication origin, a kanamycin-selectable marker, and an yfp as the reporter gene regulated by a J23100 promoter. 
     The screening assay was used to quantify the functional effects of dCpf1 mutants systematically. In AsCpf1 with altered PAM specificity, mutated 5542 and K607 residues interacted with the thymine nucleotides at the −2 and −3 PAM positions. Using the negative screening assay, we validated that the corresponding dFnCpf1 mutant (N607R/K671R) created by homologous alignment had a low activity on most expected high-GC PAM sites, and it exhibited lower PAM constrains than WT dFnCpf1. Therefore, a 750 bp DNA sequence (SEQ ID NO: 3) containing the PAM-interacting (PI) domains of dFnCpf1 mutants N607R, K671R, N607R/K671R through error-prone PCR to construct a dfncpf1 variants plasmid library. The mutagenesis library was introduced into host  E. coli  cells harboring a plasmid expressing crRNA and a plasmid carrying different SSSC PAMs upstream of the yfp gene. Expression of the dFnCpf1-crRNA system was induced using IPTG, and reduction in fluorescence was quantified to evaluate the PAM recognition and target binding efficiency of the dFnCpf1-crRNA complex ( FIG.  1   ). The invention applies the negative screening assay to impose artificial selection pressure to screen mutants that could recognize different GC-rich PAM sequence. 
     Flow cytometry was used to screen mutants with significantly reduced fluorescence as follows: The  E. coli  strain DH5a strain was used in this study.  E. coli  strain was cultured in LB (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) or M9 media (12.8 g/L Na2HPO4.7H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1.67 g/L NH4Cl, 1 mM thiamine hydrochloride, 0.4% glucose, 0.2% casamino acids, 2 mM MgSO4, 0.1 mM CaCl2). LB was used as the growth media. Cells for flow cytometric fluorescence analysis were cultured in M9 media. 
     Bacterial cells were cultured overnight, diluted 196 times using the M9 medium containing three antibiotics, and later incubated for 3 h. After incubation, cells were diluted 1000 times in the M9 medium containing three antibiotics and 200 μM IPTG followed by shaking at 37° C. for 8 h. To stop protein expression prior to flow cytometry analysis, bacterial cells were diluted using PBS containing 2 mg/mL kanamycin. The fluorescence intensity of YFP was measured using a Calibur flow cytometer (BD Biosciences, CA, USA) with appropriate settings (FSC 440, SSC 260, FITC 480). Minimum 50,000 events were collected for each sample. The geometric mean of fluorescence intensity of each sample was analyzed using FlowJo software version 7.6.2 (Treestar, USA), and the autofluorescence of  E. coli  was subtracted for each sample. The resulting dFncpf1 mutant libraries were transformed into the  E. coli  DH5a cells harboring the reporter plasmid and the crRNA plasmid. The transformants were cultured overnight (˜14 h), diluted, and induced by 200 μM IPTG for 6 h. Later, cells with relatively lower fluorescence (lower than an artificially defined threshold) were sorted into fresh LB medium using a BD Influx cell sorter (BD, USA). After 3 h of cell resuscitation, the sorted cells were plated on LB agar. The clones were picked and cultured further for flow cytometry (BD Fortessa, USA) based validation studies. Cells with relatively low fluorescence were sequenced and collected for the next mutant screening. A positive control ( E. coli  DH5a strain containing the pSC101-J23100-yfp plasmid) and negative control ( E. coli  DH5a strain containing the pSC101-J23100, pColE1-J23119-crRNA, and p15A-pTac-dfncpf1 plasmids) were used to set the appropriate gain for the fluorescence channel. 
     Directed evolution was employed to evolve the dFnCpf1 for binding to each of the eight possible SSSC (S=C,G) PAM target sequences in parallel. Finally, we obtained 86 mutants that effectively recognized the corresponding GC-rich PAM sequences respectively, compared to little or no activity for WT. In the invention, dFnCpf1 mutants with strong recognition ability of PAM CCCC and PAM CCGC is obtained, and the repression fold of YFP fluorescence value can reach more than 100 times (equivalent to the binding ability of wild-type dFnCpf1 for PAM TTTC). The dFnCpf1 mutants with strong recognition ability of PAM GCCG and PAM GCGC could inhibit the YFP fluorescence value by 60 or 70 times. The mutant with obvious recognition ability of PAM CGCC, GGCC, CGGC and GGGC could suppress the YFP fluorescence value by more than 30 times. The results are showed as Table 1: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 All dFnCpf1 variants screened from the directed evolution 
               
            
           
           
               
               
            
               
                   
                 Repression fold 
               
               
                   
                   
               
            
           
           
               
            
               
                 Mutants evolved from the PAM CCCC pathway 
               
            
           
           
               
               
               
            
               
                   
                 E566V/K671R/D751G(refer to VRG) 
                 51.22 
               
               
                   
                 VRG/E635K 
                 65.20 
               
               
                   
                 VRG/N580H 
                 63.17 
               
               
                   
                 VRG/N508H/F570L 
                 81.92 
               
               
                   
                 VRG/N508H/F570L/E752V 
                 91.30 
               
               
                   
                 VRG/N508H/F570L/N637S 
                 116.43 
               
               
                   
                 VRG/N508H/F570L/N553D 
                 102.18 
               
               
                   
                 VRG/N508H/F570L/I542V 
                 93.26 
               
               
                   
                 VRG/N508H/F570L/I542V/E559G 
                 102.77 
               
            
           
           
               
            
               
                 Mutants evolved from the PAM GCCC pathway 
               
            
           
           
               
               
               
            
               
                   
                 VRG 
                 20.34 
               
               
                   
                 VRG/F570L 
                 37.60 
               
               
                   
                 VRG/E635K 
                 37.53 
               
               
                   
                 VRG/F570L/E756G 
                 36.08 
               
               
                   
                 VRG/F570L/N634D 
                 40.90 
               
               
                   
                 VRG/F570L/D687N 
                 57.09 
               
               
                   
                 VRG/F570L/N634D/K611R 
                 61.30 
               
               
                   
                 VRG/F570L/N634D/R755K 
                 61.84 
               
               
                   
                 VRG/F570L/N634D/E767G 
                 54.26 
               
            
           
           
               
            
               
                 Mutants evolved from the PAM CCGC pathway 
               
            
           
           
               
               
               
            
               
                   
                 VRG/S518G 
                 155.27 
               
               
                   
                 VRG/S546R 
                 143.85 
               
               
                   
                 VRG/L537I 
                 33.01 
               
               
                   
                 VRG/S518G/K639R 
                 159.86 
               
               
                   
                 VRG/S518G/K639R/K719R 
                 135.95 
               
               
                   
                 VRG/L537I/K639R/F565L 
                 140.00 
               
               
                   
                 VRG/L537I/F565L/K639R/K719R 
                 143.32 
               
               
                   
                 VRG/L537I/F565L/N634D/K719R 
                 151.20 
               
            
           
           
               
            
               
                 Mutants evolved from the PAM GCGC pathway 
               
            
           
           
               
               
               
            
               
                   
                 VRG/F570L 
                 68.77 
               
               
                   
                 VRG/S546R 
                 62.69 
               
               
                   
                 VRG/F570L/E686D 
                 77.17 
               
               
                   
                 VRG/F570L/N634D/E686D 
                 68.68 
               
               
                   
                 VRG/F570L/I575M/E686D 
                 63.22 
               
               
                   
                 VRG/F570L/N634D/L572I 
                 63.23 
               
               
                   
                 VRG/F570L/N634D/F596Y 
                 68.05 
               
            
           
           
               
            
               
                 Mutants evolved from the PAM CGCC pathway 
               
            
           
           
               
               
               
            
               
                   
                 VRG/K613N (refer toVRGN) 
                 10.87 
               
               
                   
                 VRGN/F570L 
                 27.31 
               
               
                   
                 VRGN/F570L/N637S 
                 23.25 
               
               
                   
                 VRGN/N534K/N637S 
                 20.39 
               
               
                   
                 VRGN/N637S/N534K/G664V 
                 64.75 
               
               
                   
                 VRGN/N637S/N534K/G664S 
                 38.40 
               
            
           
           
               
            
               
                 Mutants evolved from the PAM GGCC pathway 
               
            
           
           
               
               
               
            
               
                   
                 VRGN/F570L/G664S 
                 35.06 
               
               
                   
                 VRGN/F570L/G664S/N637Y 
                 62.14 
               
               
                   
                 VRGN/F570L/G664S/F710S 
                 29.30 
               
               
                   
                 VRGN/F570L/G664S/K647R/I728L 
                 40.78 
               
               
                   
                 VRGN/F570L/G664S/A619V/S729R 
                 36.03 
               
            
           
           
               
            
               
                 Mutants evolved from the PAM CGGC pathway 
               
            
           
           
               
               
               
            
               
                   
                 VRGN/Y724C 
                 32.83 
               
               
                   
                 VRGN/Y724C/F570L 
                 47.10 
               
               
                   
                 VRGN/Y724C/D522V/K650R 
                 33.19 
               
               
                   
                 VRGN/Y724C/D616N 
                 33.22 
               
               
                   
                 VRGN/Y724C/K505N/F570L 
                 42.95 
               
               
                   
                 VRGN/Y724C/F570L/I582V 
                 42.26 
               
               
                   
                 VRGN/Y724C/F570L/E715D 
                 38.75 
               
            
           
           
               
            
               
                 Mutants evolved from the PAM GGGC pathway 
               
            
           
           
               
               
               
            
               
                   
                 VRGN/Y724C 
                 15.37 
               
               
                   
                 VRGN/Y724C/Q509R 
                 32.09 
               
               
                   
                 VRGN/Y724C/F570L 
                 36.37 
               
               
                   
                 VRGN/Y724C/F570L/R690I/L662I 
                 45.41 
               
               
                   
                 VRGN/Y724C/F570L/R690I/D625E 
                 41.59 
               
               
                   
                   
               
            
           
         
       
     
     Embodiment 2 
     1. PAM Preference Profiles Analysis 
     To determine PAM compatibilities in acquired mutants (separately evolved based on different GC-rich PAMs), eight M1 variants with the highest activity in the corresponding PAM trajectories were assessed for the global 64 PAM preferences (NNNC, the last base of PAM sequence is determined as C due to the weak preference) and later compared with WT dFnCpf1. A randomized PAM library (NNNC) was constructed through PCR and Gibson ligation. The 64 PAM plasmids were transformed separately into competent  E. coli  DH5a cells harboring dFnCpf1 mutants and crRNA plasmids. The fluorescence intensity of YFP was measured using a Calibur flow cytometer and the data was analyzed using FlowJo. The PAM preference profiles were analyzed and displayed using Matlab. 
     As expected, WT dFnCpf1 was found to be the most effective for NTTC PAMs, especially for the TTTC PAM. WT dFnCpf1 also targeted other PAM sequences, including NCTC, AATC, and TTCC, but at lower rates ( FIG.  3   , Table 2). By contrast, the variants that can recognize SCSC PAMs showed the highest activity at NCCC and NCTC PAMs, compared to little or no activity for WT ( FIG.  3   ). Surprisingly, the variants that can recognize SGSC PAMs were active at almost all 64 PAMs, especially the GC-rich PAMs, although the protein activity declined ( FIG.  3   , Table 2). We observed that the mutant dFnCpf1 (VRGN/Y724C/F570L/R690I/L662I), which was selected from the variants that could recognize GGGC PAM, had 52 effective identification tags (YFP fluorescence value&lt;200) out of all 64 NNNC sites (81.3%) (Table 2). Furthermore, the mutant could effectively recognize 28 PAMs out of the 32 (87.5%) GC-rich PAMs (two or more C/G within −2 to −4 bits of PAM sequence) (Table 2). Thus, based on these outcomes, the dFnCpf1 (VRGN/Y724C/F570L/R690I/L662I) variant, obtained in this study with a significantly expanded targeting range (approximately 10 folds), was referred to as broad-spectrum dFnCpf1 (bsdFnCpf1). Besides, the other three mutans (VRGN/N637S/N534K/G664V, VRGN/F570L/G664S/N637Y, VRGN/Y724C/F570L) that could recognize SGSC PAMshad a 5-fold wider range of PAM than the wild-type. 
     Sequences of bsdFnCpf1 is provided as follows (SEQ ID NO:4): 
     
       
         
           
               
               
            
               
                 ATGTCAATTTATCAAGAATTTGTTAATAAATATAGTTTAAGTAAAACTCTAAGATTTG 
                   
               
               
                   
               
               
                 AGTTAATCCCACAGGGTAAAACACTTGAAAACATAAAAGCAAGAGGTTTGATTTT 
               
               
                   
               
               
                 AGATGATGAGAAAAGAGCTAAAGACTACAAAAAGGCTAAACAAATAATTGATAAA 
               
               
                   
               
               
                 TATCATCAGTTTTTTATAGAGGAGATATTAAGTTCGGTTTGTATTAGCGAAGATTTAT 
               
               
                   
               
               
                 TACAAAACTATTCTGATGTTTATTTTAAACTTAAAAAGAGTGATGATGATAATCTAC 
               
               
                   
               
               
                 AAAAAGATTTTAAAAGTGCAAAAGATACGATAAAGAAACAAATATCTGAATATATA 
               
               
                   
               
               
                 AAGGACTCAGAGAAATTTAAGAATTTGTTTAATCAAAACCTTATCGATGCTAAAAA 
               
               
                   
               
               
                 AGGGCAAGAGTCAGATTTAATTCTATGGCTAAAGCAATCTAAGGATAATGGTATAG 
               
               
                   
               
               
                 AACTATTTAAAGCCAATAGTGATATCACAGATATAGATGAGGCGTTAGAAATAATCA 
               
               
                   
               
               
                 AATCTTTTAAAGGTTGGACAACTTATTTTAAGGGTTTTCATGAAAATAGAAAAAAT 
               
               
                   
               
               
                 GTTTATAGTAGCAATGATATTCCTACATCTATTATTTATAGGATAGTAGATGATAATTT 
               
               
                   
               
               
                 GCCTAAATTTCTAGAAAATAAAGCTAAGTATGAGAGTTTAAAAGACAAAGCTCCA 
               
               
                   
               
               
                 GAAGCTATAAACTATGAACAAATTAAAAAAGATTTGGCAGAAGAGCTAACCTTTG 
               
               
                   
               
               
                 ATATTGACTACAAAACATCTGAAGTTAATCAAAGAGTTTTTTCACTTGATGAAGTT 
               
               
                   
               
               
                 TTTGAGATAGCAAACTTTAATAATTATCTAAATCAAAGTGGTATTACTAAATTTAATA 
               
               
                   
               
               
                 CTATTATTGGTGGTAAATTTGTAAATGGTGAAAATACAAAGAGAAAAGGTATAAAT 
               
               
                   
               
               
                 GAATATATAAATCTATACTCACAGCAAATAAATGATAAAACACTCAAAAAATATAAA 
               
               
                   
               
               
                 ATGAGTGTTTTATTTAAGCAAATTTTAAGTGATACAGAATCTAAATCTTTTGTAATT 
               
               
                   
               
               
                 GATAAGTTAGAAGATGATAGTGATGTAGTTACAACGATGCAAAGTTTTTATGAGCA 
               
               
                   
               
               
                 AATAGCAGCTTTTAAAACAGTAGAAGAAAAATCTATTAAAGAAACACTATCTTTAT 
               
               
                   
               
               
                 TATTTGATGATTTAAAAGCTCAAAAACTTGATTTGAGTAAAATTTATTTTAAAAATG 
               
               
                   
               
               
                 ATAAATCTCTTACTGATCTATCACAACAAGTTTTTGATGATTATAGTGTTATTGGTAC 
               
               
                   
               
               
                 AGCGGTACTAGAATATATAACTCAACAAATAGCACCTAAAAATCTTGATAACCCTA 
               
               
                   
               
               
                 GTAAGAAAGAGCAAGAATTAATAGCCAAAAAAACTGAAAAAGCAAAATACTTATC 
               
               
                   
               
               
                 TCTAGAAACTATAAAGCTTGCCTTAGAAGAATTTAATAAGCATAGAGATATAGATAA 
               
               
                   
               
               
                 ACAGTGTAGGTTTGAAGAAATACTTGCAAACTTTGCGGCTATTCCGATGATATTTG 
               
               
                   
               
               
                 ATGAAATAGCTCAAAACAAAGACAATTTGGCACAGATATCTATCAAATATCAAAAT 
               
               
                   
               
               
                 CAAGGTAAAAAAGACCTACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCTATCA 
               
               
                   
               
               
                 AGGATCTTTTAGATCAAACTAATAATCTCTTACATAAACTAAAAATATTTCATATTAG 
               
               
                   
               
               
                 TCAGTCAGAAGATAAGGCAAATATTTTAGACAAGGATGAGCATTTTTATCTAGTATT 
               
               
                   
               
               
                 TGTGGAGTGCTACCTTGAGCTAGCGAATATAGTGCCTCTTTATAACAAAATTAGAA 
               
               
                   
               
               
                 ACTATATAACTCAAAAGCCATATAGTGATGAGAAATTTAAGCTCAATTTTGAGAACT 
               
               
                   
               
               
                 CGACTTTGGCTAATGGTTGGGATAAAAATAATGAGCCTGACAATACGGCAATTTTA 
               
               
                   
               
               
                 TTTATCAAAGATGATAAATATTATCTGGGTGTGATGAATAAGAAAAATAACAAAATA 
               
               
                   
               
               
                 TTTGATGATAAAGCTATCAAAGAAAATAAAGGCGAGGGTTATAAAAAAATTGTTTA 
               
               
                   
               
               
                 TAAACTTATACCTGGCGCAAATAAAATGTTACCTCGTGTTTTCTTTTCTGCTAAATC 
               
               
                   
               
               
                 TATAAAATTTTATAATCCTAGTGAAGATATACTTATAATAAGAAATCATTCCACACAT 
               
               
                   
               
               
                 ACAAAAAATGGTAGTCCTCAAAAAGGATATGAAAAATTTGAGTTTAATATTGAAGA 
               
               
                   
               
               
                 TTGCCGAAAATTTATAGATTTTTGTAAACAGTCTATAAGTAAGCATCCGGAGTGGA 
               
               
                   
               
               
                 AAGATTTTGGATTTAGATTTTCTGATACTCAAAGATATAATTCTATAGGTGAATTTTA 
               
               
                   
               
               
                 TAGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATATCAGAGAGC 
               
               
                   
               
               
                 TATATTGATAGCGTAGTTAATCAGGGTAAATTGTACCTATTCCAAATCTATAATAAAG 
               
               
                   
               
               
                 ATTTTTCAGCTTATAGCAAAGGGCGACCAAATCTACATACTTTATATTGGAAAGCGC 
               
               
                   
               
               
                 TGTTTGATGAGAGAAATCTTCAAGATGTGGTTTATAAGCTAAATGGTGAGGCAGAG 
               
               
                   
               
               
                 CTTTTTTATCGTAAACAATCAATACCTAAAAAAATCACTCACCCAGCTAAAGAGGC 
               
               
                   
               
               
                 AATAGCTAATAAAAACAAAGATAATCCTAAAAAAGAGAGTGTTTTTGAATATGATT 
               
               
                   
               
               
                 TAATCAAAGATAAACGCTTTACTGAAGATAAGTTTTTCTTTCACTGTCCTATTACAA 
               
               
                   
               
               
                 TCAATTTTAAATCTAGTGGAGCTAATAAGTTTAATGATGAAATCAATTTATTGCTAA 
               
               
                   
               
               
                 AAGAAAAAGCAAATGATGTTCATATATTAAGTATAGCAAGAGGTGAAAGACATTTA 
               
               
                   
               
               
                 GCTTACTATACTTTGGTAGATGGTAAAGGCAATATCATCAAACAAGATACTTTCAAC 
               
               
                   
               
               
                 ATCATTGGTAATGATAGAATGAAAACAAACTACCATGATAAGCTTGCTGCAATAGA 
               
               
                   
               
               
                 GAAAGATAGGGATTCAGCTAGGAAAGACTGGAAAAAGATAAATAACATCAAAGA 
               
               
                   
               
               
                 GATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAAATAGCTAAGCTAGTTATAG 
               
               
                   
               
               
                 AGTATAATGCTATTGTGGTTTTTGAGGATTTAAATTTTGGATTTAAAAGAGGGCGTT 
               
               
                   
               
               
                 TCAAGGTAGAGAAGCAGGTCTATCAAAAGTTAGAAAAAATGCTAATTGAGAAACT 
               
               
                   
               
               
                 AAACTATCTAGTTTTCAAAGATAATGAGTTTGATAAAACTGGGGGAGTGCTTAGAG 
               
               
                   
               
               
                 CTTATCAGCTAACAGCACCTTTTGAGACTTTTAAAAAGATGGGTAAACAAACAGG 
               
               
                   
               
               
                 TATTATCTACTATGTACCAGCTGGTTTTACTTCAAAAATTTGTCCTGTAACTGGTTTT 
               
               
                   
               
               
                 GTAAATCAGTTATATCCTAAGTATGAAAGTGTCAGCAAATCTCAAGAGTTCTTTAGT 
               
               
                   
               
               
                 AAGTTTGACAAGATTTGTTATAACCTTGATAAGGGCTATTTTGAGTTTAGTTTTGAT 
               
               
                   
               
               
                 TATAAAAACTTTGGTGACAAGGCTGCCAAAGGCAAGTGGACTATAGCTAGCTTTG 
               
               
                   
               
               
                 GGAGTAGATTGATTAACTTTAGAAATTCAGATAAAAATCATAATTGGGATACTCGA 
               
               
                   
               
               
                 GAAGTTTATCCAACTAAAGAGTTGGAGAAATTGCTAAAAGATTATTCTATCGAATAT 
               
               
                   
               
               
                 GGGCATGGCGAATGTATCAAAGCAGCTATTTGCGGTGAGAGCGACAAAAAGTTTT 
               
               
                   
               
               
                 TTGCTAAGCTAACTAGTGTCCTAAATACTATCTTACAAATGCGTAACTCAAAAACA 
               
               
                   
               
               
                 GGTACTGAGTTAGATTATCTAATTTCACCAGTAGCAGATGTAAATGGCAATTTCTTT 
               
               
                   
               
               
                 GATTCGCGACAGGCGCCAAAAAATATGCCTCAAGATGCTGATGCCAATGGTGCTTA 
               
               
                   
               
               
                 TCATATTGGGCTAAAAGGTCTGATGCTACTAGGTAGGATCAAAAATAATCAAGAGG 
               
               
                   
               
               
                 GCAAAAAACTCAATTTGGTTATCAAAAATGAAGAGTATTTTGAGTTCGTGCAGAAT 
               
               
                   
               
               
                 AGGAATAAC. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 PAM preference analysis profile data.YFP fluorescence intensity after 
               
               
                 200 μM IPTG induction was used as the characterization value. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 GCCC-M1 
                   
                   
               
               
                   
                   
                 CCCC-M1 
                 (VRG/F570L/ 
                 CCGC-M1 
                 GCGC-M1 
               
               
                 PAM 
                 WT 
                 (VRG/N508H/N637S) 
                 N634D/R755K) 
                 (VRG/S518G/K639R) 
                 (VRG/F570L/E686D) 
               
               
                   
               
               
                 AAAV 
                 3839.2 
                 3921.2 
                 3606.6 
                 4136 
                 3960.8 
               
               
                 ATAV 
                 2832.4 
                 989.4 
                 670.8 
                 1287.2 
                 1092.6 
               
               
                 ACAV 
                 2891.4 
                 251.4 
                 199.8 
                 425.8 
                 318.2 
               
               
                 AGAV 
                 4170.4 
                 4149.2 
                 3872.4 
                 4457.8 
                 4328.6 
               
               
                 TAAV 
                 3818.4 
                 4167.4 
                 3193.8 
                 4063.2 
                 3946.6 
               
               
                 TTAV 
                 1284.6 
                 217.6 
                 140.4 
                 274 
                 203.4 
               
               
                 TCAV 
                 3186.8 
                 404.8 
                 252.6 
                 489.2 
                 378.2 
               
               
                 TGAV 
                 3927 
                 4490.4 
                 4171.6 
                 4264.4 
                 4294.8 
               
               
                 CAAV 
                 4158 
                 4275.6 
                 3535.6 
                 4252.4 
                 4145.6 
               
               
                 CTAV 
                 1851.8 
                 262.8 
                 172.4 
                 393 
                 292 
               
               
                 CCAV 
                 3599 
                 306.2 
                 182.8 
                 387.8 
                 291.4 
               
               
                 CGAV 
                 4281.2 
                 4429.4 
                 3865.2 
                 4561.4 
                 4219.4 
               
               
                 GAAV 
                 4430.4 
                 4945.2 
                 4512 
                 4609 
                 4669.6 
               
               
                 GTAV 
                 2943.8 
                 817 
                 471.8 
                 1433 
                 1600.2 
               
               
                 GCAV 
                 2010.8 
                 146 
                 93.06 
                 209.4 
                 153.6 
               
               
                 GGAV 
                 4396 
                 4816.8 
                 4538.8 
                 4545.4 
                 4584.4 
               
               
                 AATV 
                 53.32 
                 9.72 
                 7.1 
                 18 
                 8.64 
               
               
                 ATTV 
                 31.78 
                 33.92 
                 30.02 
                 41.46 
                 34.42 
               
               
                 ACTV 
                 115.8 
                 31.22 
                 21.84 
                 32.56 
                 26.5 
               
               
                 AGTV 
                 2956.2 
                 2154 
                 1173.2 
                 2302.4 
                 2418 
               
               
                 TATV 
                 2113.2 
                 965.4 
                 516.8 
                 1258 
                 1004 
               
               
                 TTTV 
                 9.56 
                 19.04 
                 14.56 
                 36.7 
                 16.46 
               
               
                 TCTV 
                 39.56 
                 22.94 
                 16.22 
                 18.74 
                 16.82 
               
               
                 TGTV 
                 2064.4 
                 1902.2 
                 1507.4 
                 2330.6 
                 2227 
               
               
                 CATV 
                 3298.4 
                 1272.4 
                 694 
                 1643.6 
                 1534.8 
               
               
                 CTTV 
                 24.04 
                 41.24 
                 33.38 
                 55.62 
                 37.08 
               
               
                 CCTV 
                 130 
                 32.96 
                 29.92 
                 42.06 
                 32.62 
               
               
                 CGTV 
                 3404.2 
                 2714.6 
                 2040 
                 3260 
                 3029.6 
               
               
                 GATV 
                 2673.6 
                 982.2 
                 612.6 
                 1516.4 
                 1387.8 
               
               
                 GTTV 
                 16.3 
                 33.16 
                 23.68 
                 42.02 
                 36.44 
               
               
                 GCTV 
                 74.1 
                 25.28 
                 24.04 
                 30.72 
                 26.34 
               
               
                 GGTV 
                 2458.8 
                 3046.6 
                 2279.2 
                 3140.4 
                 3041.2 
               
               
                 AACV 
                 3759.6 
                 755.4 
                 350.6 
                 988.8 
                 758.2 
               
               
                 ATCV 
                 1551.6 
                 322.2 
                 155 
                 379 
                 258 
               
               
                 ACCV 
                 912.6 
                 43.26 
                 40.5 
                 71.76 
                 50.56 
               
               
                 AGCV 
                 3878.4 
                 3129.4 
                 2253.6 
                 3450.8 
                 3125.6 
               
               
                 TACV 
                 3527.6 
                 1287.6 
                 733.4 
                 1640.6 
                 1607.4 
               
               
                 TTCV 
                 50.1 
                 32.42 
                 26.3 
                 49.24 
                 33.06 
               
               
                 TCCV 
                 186.4 
                 16.62 
                 14.8 
                 21.5 
                 16.94 
               
               
                 TGCV 
                 4192.6 
                 4230.4 
                 3545.6 
                 3401.6 
                 4312 
               
               
                 CACV 
                 3403.4 
                 537.8 
                 236.6 
                 787.6 
                 516.2 
               
               
                 CTCV 
                 303.6 
                 76.36 
                 57.4 
                 113.4 
                 85.08 
               
               
                 CCCV 
                 816 
                 37.14 
                 30.4 
                 51.68 
                 37.68 
               
               
                 CGCV 
                 4079.2 
                 3433.8 
                 2528.6 
                 3728.6 
                 3042.6 
               
               
                 GACV 
                 3428.6 
                 876.4 
                 447.6 
                 1187.8 
                 888.6 
               
               
                 GTCV 
                 224 
                 108.72 
                 66.48 
                 137 
                 116.2 
               
               
                 GCCV 
                 466.2 
                 31.2 
                 27.66 
                 99.44 
                 34 
               
               
                 GGCV 
                 3518 
                 3423 
                 2746.2 
                 3416.6 
                 3416.2 
               
               
                 AAGV 
                 3882.4 
                 3735 
                 2621.6 
                 3779.8 
                 3554.4 
               
               
                 ATGV 
                 3581.8 
                 1727.8 
                 1222.2 
                 2032.2 
                 1813.8 
               
               
                 ACGV 
                 2907.8 
                 135 
                 84.62 
                 208.8 
                 141.2 
               
               
                 AGGV 
                 3815 
                 4128.8 
                 3860.2 
                 4174.6 
                 3920 
               
               
                 TAGV 
                 4256.2 
                 2930.6 
                 2195 
                 3358.6 
                 3257.2 
               
               
                 TTGV 
                 2314.8 
                 392.6 
                 360 
                 626.4 
                 493.8 
               
               
                 TCGV 
                 1421.6 
                 46.04 
                 179.46 
                 75.3 
                 59.62 
               
               
                 TGGV 
                 3952.6 
                 4305.8 
                 4220.2 
                 4268.4 
                 4209.8 
               
               
                 CAGV 
                 3824.8 
                 3586.8 
                 2651.2 
                 3710.2 
                 3661 
               
               
                 CTGV 
                 2276 
                 260 
                 154 
                 376 
                 326 
               
               
                 CCGV 
                 2731.6 
                 220.8 
                 164.6 
                 37.74 
                 69.88 
               
               
                 CGGV 
                 4255 
                 4384.4 
                 3914.2 
                 4349.4 
                 4288.8 
               
               
                 GAGV 
                 4204.2 
                 3954.8 
                 3281.2 
                 4179.4 
                 4135.6 
               
               
                 GTGV 
                 1814.2 
                 268 
                 228 
                 451.4 
                 356.4 
               
               
                 GCGV 
                 1769.8 
                 44.76 
                 53.5 
                 96.02 
                 68.16 
               
               
                 GGGV 
                 3845.5 
                 4366.6 
                 4116.6 
                 4299.4 
                 4276.6 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 CGCC-M1 
                 GGCC-M1 
                   
                 GGGC-M1 
               
               
                   
                 (VRGN/N637S/ 
                 (VRGN/F570L/ 
                 CGGC-M1 
                 (VRGN/Y724C/F570L/ 
               
               
                 PAM 
                 N534K/G664V) 
                 G664S/N637Y) 
                 (VRGN/Y724C/F570L) 
                 R690I/L662I) 
               
               
                   
               
               
                 AAAV 
                 2510.88 
                 3513.4 
                 2033 
                 2045 
               
               
                 ATAV 
                 164.80 
                 508.2 
                 233.4 
                 176.8 
               
               
                 ACAV 
                 327.60 
                 1224 
                 386.6 
                 359 
               
               
                 AGAV 
                 212.20 
                 594.2 
                 302.4 
                 196 
               
               
                 TAAV 
                 246.60 
                 767 
                 506 
                 390.2 
               
               
                 TTAV 
                 32.10 
                 57.5 
                 33.12 
                 30.26 
               
               
                 TCAV 
                 1343.20 
                 2327.4 
                 345.2 
                 337 
               
               
                 TGAV 
                 136.80 
                 361.6 
                 139.8 
                 123.4 
               
               
                 CAAV 
                 1113.33 
                 2816.4 
                 1160.6 
                 1296.8 
               
               
                 CTAV 
                 34.76 
                 57.5 
                 35.46 
                 27.32 
               
               
                 CCAV 
                 863.00 
                 1611.6 
                 200 
                 159 
               
               
                 CGAV 
                 85.14 
                 136.8 
                 64.56 
                 63.58 
               
               
                 GAAV 
                 3166.40 
                 4037.4 
                 2040.6 
                 1605.2 
               
               
                 GTAV 
                 710.60 
                 673.8 
                 114 
                 125.8 
               
               
                 GCAV 
                 118.20 
                 260.2 
                 103 
                 93.48 
               
               
                 GGAV 
                 806.60 
                 1350.6 
                 296.2 
                 239 
               
               
                 AATV 
                 9.70 
                 11.62 
                 10.1 
                 5.62 
               
               
                 ATTV 
                 39.18 
                 36.62 
                 38.26 
                 26.22 
               
               
                 ACTV 
                 67.82 
                 81.28 
                 61.6 
                 49.16 
               
               
                 AGTV 
                 82.08 
                 88.56 
                 74.68 
                 51.16 
               
               
                 TATV 
                 32.70 
                 36.06 
                 47.28 
                 32.92 
               
               
                 TTTV 
                 19.26 
                 16.14 
                 17.26 
                 14.38 
               
               
                 TCTV 
                 35.06 
                 28.38 
                 28 
                 20.92 
               
               
                 TGTV 
                 66.53 
                 71.04 
                 72.66 
                 46.4 
               
               
                 CATV 
                 47.68 
                 48.96 
                 60.8 
                 34.94 
               
               
                 CTTV 
                 54.80 
                 48.14 
                 27.36 
                 30.36 
               
               
                 CCTV 
                 82.72 
                 125.2 
                 65.92 
                 66.42 
               
               
                 CGTV 
                 64.56 
                 71.82 
                 63.86 
                 42.58 
               
               
                 GATV 
                 75.42 
                 143.6 
                 96.14 
                 93.44 
               
               
                 GTTV 
                 33.46 
                 29.86 
                 36.6 
                 23.94 
               
               
                 GCTV 
                 38.00 
                 39.98 
                 41.66 
                 30.62 
               
               
                 GGTV 
                 71.16 
                 81.08 
                 84.8 
                 53.56 
               
               
                 AACV 
                 88.08 
                 154 
                 135.4 
                 75.42 
               
               
                 ATCV 
                 109.92 
                 148.2 
                 173 
                 88.66 
               
               
                 ACCV 
                 150.60 
                 144 
                 164.8 
                 102.4 
               
               
                 AGCV 
                 117.00 
                 153.4 
                 168.4 
                 104.6 
               
               
                 TACV 
                 157.40 
                 320 
                 207.8 
                 254.6 
               
               
                 TTCV 
                 36.28 
                 31.2 
                 34.14 
                 26.32 
               
               
                 TCCV 
                 34.16 
                 32.12 
                 35.42 
                 30.56 
               
               
                 TGCV 
                 116.14 
                 127.4 
                 119.4 
                 113 
               
               
                 CACV 
                 150.80 
                 237 
                 65.12 
                 58.54 
               
               
                 CTCV 
                 37.16 
                 36.12 
                 44.12 
                 32.72 
               
               
                 CCCV 
                 128.00 
                 170 
                 133.2 
                 141.2 
               
               
                 CGCV 
                 69.64 
                 84.72 
                 98.8 
                 67.08 
               
               
                 GACV 
                 46.74 
                 59.4 
                 82.2 
                 53.28 
               
               
                 GTCV 
                 56.98 
                 72.36 
                 93.12 
                 54.84 
               
               
                 GCCV 
                 57.66 
                 58.96 
                 70.4 
                 46.44 
               
               
                 GGCV 
                 100.63 
                 99.8 
                 121.2 
                 80.42 
               
               
                 AAGV 
                 2551.80 
                 3412.8 
                 993.4 
                 673.6 
               
               
                 ATGV 
                 1331.00 
                 2340.8 
                 1357 
                 1442.6 
               
               
                 ACGV 
                 177.60 
                 341 
                 210.6 
                 152.2 
               
               
                 AGGV 
                 2070.00 
                 3179.4 
                 1979.2 
                 1812 
               
               
                 TAGV 
                 220.40 
                 615.8 
                 281.8 
                 183.2 
               
               
                 TTGV 
                 483.80 
                 667.8 
                 165.6 
                 155 
               
               
                 TCGV 
                 68.92 
                 109.6 
                 73.04 
                 44.4 
               
               
                 TGGV 
                 140.20 
                 265.4 
                 148.2 
                 101.6 
               
               
                 CAGV 
                 1877.40 
                 2750.6 
                 577.4 
                 512 
               
               
                 CTGV 
                 41.36 
                 48 
                 35.6 
                 26.4 
               
               
                 CCGV 
                 17.85 
                 979.2 
                 45.12 
                 6.38 
               
               
                 CGGV 
                 102.94 
                 144.2 
                 90.14 
                 62.66 
               
               
                 GAGV 
                 1309.60 
                 2915.4 
                 1528 
                 1560.2 
               
               
                 GTGV 
                 79.08 
                 84.6 
                 64.8 
                 45.42 
               
               
                 GCGV 
                 81.66 
                 127.2 
                 75.46 
                 54.4 
               
               
                 GGGV 
                 141.40 
                 204.4 
                 146.2 
                 121.6 
               
               
                   
               
            
           
         
       
     
     Embodiment 3 
     The dfncpf1 gene in the screening system was replaced with apobec1-dfncpf1-ugi gene (for expressing base editor dFnCpf1-BE) or apobec1-bsdfncpf1-ugi gene (for expressing base editor bsdFnCpf1-BE). The base editing efficiency was verified in  Escherichia coli . The ugi and apobec1 genes were synthesized by Genscript Inc. After 48 hours of IPTG induction, bacteria were collected to extract plasmids, and primers were designed to build a library for next-generation sequencing. 
     The statistical results of next-generation sequencing are shown in  FIG.  4   . As expected, bsdFnCpf1-BE exhibited substantially improved C to T editing efficiency across all the 15 PAMs ( FIG.  4   ). Compared with the wild-type base editor dFnCpf1-BE, bsdFnCpf1-BE has higher editing efficiency and wider PAM selectivity. These data validated the greatly improved PAM recognition range of bsdFnCpf1, which enabled bsdFnCpf1 to target more gene sequences.