Construction Of Genome Edited Mouse Disease Model Using AsCas12f1 System With Engineered SgRNA

CRISPR-Cas system has become a mainstream technology for gene editing due to its simplicity and efficiency,especially Cas9 and Cas12 a systems.However,they typically require dual AAV vectors for in vivo administration due to their own high molecular weight,which significantly lowers the in vivo delivery effectiveness of gene editors.To address this issue,tiny Cas nuclease research and development are desperately needed.Cas12 f nuclease,which belongs to the V-F subtype effector protein in the class 2 CRISPR-Cas system,is the most compact CRISPR effector nuclease found in the current study.Its size is only one-third the size of Sp Cas9 and Cas12 a nuclease,and thus has great potential for clinical therapeutic applications.Among them,the compact AsCas12f1 protein from Acidibacillus sulfurooxidans,which consists of422 amino acids,can cleave double-stranded DNA under RNA guidance via a PAM that recognises NTTR(where R stands for either A or G).However,due to its low editing efficiency in mammalian cells,it greatly However,its low editing efficiency in mammalian cells greatly limits its application in the field of gene editing.Un1Cas12f1 and Spa Cas12f1 gene editing systems,which belong to the same family as AsCas12f1,have successfully improved their editing efficiency in cells by optimizing sg RNA(tracr RNA-cr RNA).Among them,Un1Cas12f1 has successfully obtained efficient guided g RNA by systematically designing five engineered g RNA strategies.Thus,the genome editing efficiency of CRISPR-Un1Cas12f1 in mammalian cell,which was comparable to Sp Cas9.Therefore,this study aimed to systematically optimize the sg RNA of the compact AsCas12f1 gene editing system to improve its editing efficiency in cells and animal embryos,and to construct animal models mimicking human genetic diseases using the AsCas12f1 system.Firstly,in order to validate the gene editing efficiency of the natural AsCas12f1 system on cells,15 target sites were selected in this study on HEK293 T cells,which contained TTTG/TTTA/CTTG/ATTG types of PAMs.The editing efficiencies of AsCas12f1 system at the 15 target sites did not exceed 10%.The highest insertion or deletion(Indel)was the PDCD1 site,with a mutation efficiency of 9.31%.It indicated that the overall gene editing efficiency of the natural AsCas12f1 system was low,and further development was urgently needed to enhance editing efficiency.In order to effectively improve the editing efficiency of the AsCas12f1 system,the sg RNA scaffold was optimized as follows: modified sg RNA scaffold(T4AT6/GGG);adjust the binding length(MS)of cr RNA and tracr RNA;the disordered region(DS)at the stem loop of g RNA was deleted.In addition,combining the above efficiency-enhancing schemes were combined to obtain superior sg RNAs.The superior sg RNA version(As-v1)was combined by T4AT6 and MS.Then it was combined with AsCas12f1 protein to form a new engineering system named AsCas12f1-v1.Finally,we compared the gene editing efficiency of the AsCas12f1-v1 system with the AsCas12f1 system and Un1Cas12f1 system in HEK293 T cells.The results showed that the editing efficiency of AsCas12f1 system optimized by sg RNA was significantly improved in HEK293 T cells.Secondly,in order to further verify the effectiveness of the optimized AsCas12f1 system gene editing,the efficiency of the optimized AsCas12f1 system was verified on mouse embryos and rabbit embryos,respectively.We first selected Dctn6,Komp,Tyr,Dmd and Polg five target sites on mouse embryos.And then the m RNA of AsCas12f1 and the corresponding As-v1 mixed system were injected into mouse embryonic cells by microinjection.Finally,the blastocyst cells were collected to identify the gene editing efficiency.The results of mouse embryos showed that the optimized AsCas12f1 system successfully completed gene editing at five sites of mouse embryos with editing efficiencies of 15.62 %-62.47 % compared to the AsCas12f1 system.Similarly,we also selected five gene loci of Hbb,Fgf5,Wrn,Otc and Mstn in rabbit embryos.The results of rabbit embryos showed that the optimized AsCas12f1 system completed gene editing at five sites of rabbit embryos,and the editing efficiency was 4.75 %-16.82 %Finally,in order to verify that the optimized AsCas12f1 system can not only successfully develop gene editing in vitro but also complete effective editing in vivo.We transferred the microinjected fertilized ovum containing the optimized AsCas12f1 m RNA and Tyr,Dmd,Foxp1 g RNA into the fallopian tubes on both sides of the surrogate mother.After the mice were born for 7-10 d,the mice were labelled and the genomes were extracted for sequencing and identification.The sequencing results showed that the AsCas12f1-v1 system developed gene editing in mice,and the editing efficiency was Tyr(38.87 %-49.66 %),Dmd(33.47 %-47.16 %),Foxp1(23.41 %-37.42 %).In addition,we performed preliminary phenotypic characterisation of Tyr mutant mice,Dmd mutant mice and Foxp1 mutant mice.The results showed that all three mouse mutation models successfully simulated the typical phenotypes of corresponding human diseases.In summary,this study effectively improved the editing efficiency of AsCas12f1 system in cells and animal embryos by optimizing its sg RNA.The engineered AsCas12f1 system was used to complete in vivo editing in mice and construct mouse models simulating human disease mutations.This study not only provides a theoretical basis for the optimization of compact gene editing systems,but also provides new gene editing tools for constructing animal models of human genetic diseases.