New therapeutic strategies for the rapid and effective treatment of drug-resistant tuberculosis are highly desirable,and their development can be drastically accelerated by facile genetic manipulation methods in Mycobacterium tuberculosis(M.tuberculosis).Clustered regularly interspaced short palindromic repeat(CRISPR)base editors allow for rapid,robust,and programmed single-base substitutions and gene inac-tivation,yet no such systems are currently available in M.tuberculosis.By screening distinct CRISPR base editors,we discovered that only the unusual Streptococcus thermophilus CRISPR associated protein 9(St1Cas9)cytosine base editor(CBE)-but not the widely used Streptococcus pyogenes Cas9(SpCas9)or Lachnospiraceae bacterium Cpf1(LbCpf1)CBEs-is active in mycobacteria.Despite the notable C-to-T con-versions,a high proportion of undesired byproducts exists with St1Cas9 CBE.We therefore engineered St1Cas9 CBE by means of uracil DNA glycosylase inhibitor(UGI)or uracil DNA glycosylase(UNG)fusion,yielding two new base editors(CTBE and CGBE)capable of C-to-T or C-to-G conversions with dramatically enhanced editing product purity and multiplexed editing capacity in Mycobacterium smegmatis(M.smeg-matis).Because wild-type St1Cas9 recognizes a relatively strict protospacer adjacent motif(PAM)sequence for DNA targeting,we engineered a PAM-expanded St1Cas9 variant by means of structure-guided protein engineering for the base editors,substantially broadening the targeting scope.We first developed and characterized CTBE and CGBE in M.smegmatis,and then applied CTBE for genome editing in M.tuberculosis.Our approaches significantly reduce the efforts and time needed for precise genetic manipulation and will facilitate functional genomics,antibiotic-resistant mechanism study,and drug-target exploration in M.tuberculosis and related organisms.

Hongyuan Zhang;Yifei Zhang;Wei-Xiao Wang;Weizhong Chen;Xia Zhang;Xingxu Huang;Wei Chen;Quanjiang Ji

School of Physical Science and Technology,ShanghaiTech University,Shanghai 201210,China;University of Chinese Academy of Sciences,Beijing 100049,China;Clinical Research Center,the Second Hospital of Nanjing,Nanjing University of Chinese Medicine,Nanjing 210003,China;Department of Tuberculosis,the Second Hospital of Nanjing,Nanjing University of Chinese Medicine,Nanjing 210003,China;Gene Editing Center,School of Life Science and Technology,ShanghaiTech University,Shanghai 201210,China;Guangzhou Laboratory,Guangzhou 510120,China

工程（英文）

[1]Hongyuan Zhang;Yifei Zhang;Wei-Xiao Wang;Weizhong Chen;Xia Zhang;Xingxu Huang;Wei Chen;Quanjiang Ji-.PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G Base Editors for Programmable Base Editing in Mycobacteria)[J].工程（英文）,2022(08):67-77

Mycobacteria,St1Cas9,LbCpf1,CBEs,CTBE,CGBE

PAM,Expanded,Streptococcus,thermophilus,Base,Editors,Programmable,Editing,New,therapeutic,strategies,rapid,effective,treatment,drug,resistant,tuberculosis,are,highly,desirable,their,development,drastically,accelerated,facile,genetic,manipulation,methods,Mycobacterium,Clustered,regularly,interspaced,short,palindromic,repeat,CRISPR,base,editors,allow,robust,programmed,single,substitutions,inac,tivation,yet,such,systems,currently,available,By,screening,distinct,we,discovered,that,only,unusual,associated,protein,cytosine,but,widely,used,pyogenes,SpCas9,Lachnospiraceae,active,mycobacteria,Despite,notable,proportion,undesired,byproducts,exists,We,therefore,engineered,means,uracil,glycosylase,inhibitor,UGI,UNG,fusion,yielding,two,new,capable,conversions,dramatically,enhanced,editing,purity,multiplexed,capacity,smegmatis,Because,wild,type,recognizes,relatively,strict,protospacer,adjacent,motif,sequence,targeting,expanded,variant,structure,guided,engineering,substantially,broadening,scope,first,developed,characterized,then,applied,genome,Our,approaches,significantly,reduce,efforts,needed,precise,will,facilitate,functional,genomics,antibiotic,mechanism,study,exploration,related,organisms

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