| The rapid development of enabling technology in synthetic biology provides a large number of genetic manipulation tools for genome “rewriting”.Among them,gene editing and its derivative technology based onCRISPR play a great role in promoting the realization of this goal.Since the birth of CRISPR-Cas gene editing technology in 2012,a number of efficient and accurate gene editing tools have been developed around gene deletion,insertion and base conversion.In particular,the base editing technology developed in recent years has played an irreplaceable role in reshaping the function of life.Recently,various base editing systems based on the classic CRISPR-Cas system have been continuously created and adapted to various types of chassis cells,such as eukaryotic and prokaryotic cells.They have carried out application exploration in many important fields such as gene therapy,design of industrial strain,crop stress resistance and so on.The design and modification of industrial strains is an important application field of base editing technology.At present,there are many different types of base editing systems were applied to industrial strains metabolic regulation,functional gene modification and other aspects.In the process of application,many bottlenecks have also been found,such as low editing efficiency,narrow editing window,poor adjustability of editing efficiency and low mutation diversification.Therefore,the development of gene editing system with high,adjustable efficiency,and wide editing window will further promote the research and development of microbial synthetic biology and the rapid construction of “intelligent chassis” cells.In this study,CRISPR systems were selected as the research object.Two kinds of efficient and portable gene and base editing tools were constructed based onCRISPR-Cas system in B.subtilis.The feasibility and effectiveness of the above system in the in situ evolution of endogenous proteins and the construction of higher version chassis were also verified.The main research results are as follows:(1)Three genome editing systems based onCRISPR-Cas12 a were constructed in B.subtilis.The efficiency of these systems in the deletion of single gene,multiple gene,extremely large gene cluster,and the insertion of single gene was verified in B.subtilis.Firstly,we heterogenetically reconstructed the single plasmid editing system(All-in-one,AIO)based onCRISPR-Cas12 a in B.subtilis,and took the endogenous genes sac A,lac A and lig DV of B.subtilis as examples to investigate their deletion ability,with the efficiency of 100%,and taking the heterologous gene sfgfp as an example to investigate its insertion ability,and the efficiency was 9%.Then,a Two-Plasmids(TP)editing system based onCRISPR-Cas12 a was constructed in B.subtilis.Using this system to edit genes sac A and lac A,the results showed that the deletion efficiency was 100% and 91%,respectively.Finally,a Chromosomally Integrated Genome Editing system(CIGE)based onCRISPR-Cas12 a was heterologous reconstructed in B.subtilis.The deletion efficiency of endogenous genes sac A,bac operon and pps operon was 100%,100% and 80%,respectively;the insertion efficiency of sfgfp is82%;and the efficiency of simultaneous deletion of sac A and apr E is 58%.The three genome editing systems can be suitable for different application circumstances,showing high editing efficiency.(2)A programmable cytosine base editing system CRISPR-CDA-nCas9-UGI was constructed and successfully applied to in situ evolution of membrane protein and protein complex.A cytosine base editor(CBE)system CRISPR-CDA-nCas9 based cytosine deaminase CDA from Petromyzon marinus(Pm CDA)and CRISPR-Cas9 from Streptococcus pyogenes(CRISPR-SpnCas9)was designed and constructed in B.subtilis,and the editing efficiency,editing window,and adjustability of editing efficiency of CRISPR-CDA-nCas9 was investigate.Through the modification of sg RNA and the addition of Uracil DNA glycosylase inhibitor(UGI),the editing efficiency of editable sites in the window was further improved and the editing window was widened.On this basis,multiplexed editing system based onCRISPR-CDA-nCas9-UGI was designed and constructed,and the adjustability of the editing efficiency for the system is verified.The membrane protein Bce B and protein complex Sec YEG were evolved in situ using CRISPR-CDA-nCas9-UGI base editing system,and mutants with significantly improved protein secretion efficiency were successfully screened.(3)A double-deaminase base editing system based onCRISPR was designed and constructed to realize in situ modification of chassis functional genes and improve the resistance of chassis to antimicrobial active peptides.The programmable double-deaminase base editing system CRISPR-ABE8e-CDA-nCas9 based on Petromyzon marinus derived cytosine deaminase CDA(Pm CDA),Escherichia coli derived mutant adenine deaminase ABE8 e,and S.pyogenes derived CRISPR-Cas9(CRISPR-Sp Cas9)was designed and constructed in B.subtilis.sig E is used as the target gene for editing,which shows that the system has extremely high editing efficiency of A-to-G and C-to-T.The editing results of selecting bceB and pks operon as targets show that the system is universal for gene editing,and can simultaneously edit multiple genes in an 8～11 nt editing window.On this basis,the adjustability of editing efficiency for CRISPR-ABE8e-CDA-nCas9 was investigated,which is suitable to the construction of mutant library.Using the system,the mutant library of endogenous PsdB protein in B.subtilis was constructed,and the higher version chassis with increased resistance to nisin were successfully screened.(4)The recently discovered Cas12b protein was modified,and the catalytically deactivated target protein d Cas12b was successfully obtained.On this basis,CRISPRi system and base editing system were constructed.A single-plasmid gene deletion system based onCRISPR-Cas12b derived from B.hisashii was designed,and the deletion efficiency for sac A and apr E was 100%.Three key sites(D574,E828 and D952)that affect the nuclease activity of Cas12b were found through the molecular docking and first-order sequence alignment between different sources of Cas12b.The nuclease inactivation mutant was obtained by sitedirected mutation,which retained the binding ability with sg RNA.On this basis,the CRISPRi system based on d Cas12b was developed,and the level of weakening gene expression was investigated using e GFP and 6 kinds of promoters as targets.Moreover,a CBE system based on d Cas12b was designed and developed,which can realize the conversion from C to T in a wide editing window(～16 nt).In conclusion,this study takes CRISPR system as the research object,and has constructs CRISPR-Cas9,CRISPR-Cas12 a,and CRISPR-Cas12b gene and base editing systems in B.subtilis,an important industrial chassis.The problems of low editing efficiency,poor adjustability and narrow editing window of the gene editing system in the bacterial chassis have been successfully solved.In addition,the BE system has the features of adjustable editing efficiency and wide editing window,which enables large-scale genome scanning to construct diverse mutation libraries,thus accelerating the design and construction of "customized" microbial chassis. |
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