Gene Editing For G6PC3 Deficiency At Embryonic Level Therapeutic Research

To cure and prevent monogenic diseases from being passed on to offsprings thereby achieve the dream of these couples to have healthy offsprings is an urgent problem to be solved at present,which requires gene therapy at the germ cell level.G6PC3 gene is a major pathogenic gene of Severe Congenital Neutropenia type 4（SCN4）,and its disfunction mainly caused by single base mutation,which can be exactly revised by Base Editing.SCN4 will lead to neutropenia and dysfunction,which carries high risk of serious infection even involving heart or urinary,and reproductive system abnormalities.The new technology of base editing is being used to address genetic illnesses.Base editing methods can be used to fix the harmful mutation,restoring the encoded protein’s normal function and achieving the goal of treating genetic illnesses.The third generation of base editing technology,known as Clustered Regularly Interspaced Short Palindromic Repeats/crispr-associated proteins（CRISPR/Cas）,is one of them and is extensively employed in biological research.For the precise editing of a single base,the Base Editor（BE）based on the CRISPR/Cas system is used.It can achieve the goal of gene editing by using the Cas9 nicking enzyme and matching deaminase to edit specific bases,mediating the conversion of pyrimidine to pyrimidine and purine to purine without producing DNA double-strand breaks.Base editing technology has been extensively employed in developing superior kinds and creating disease models,albeit being a long way from therapeutic application.We found a case of SCN4 in the clinic.Two heterozygous mutations were found after whole blood was collected for gene detection:c.295C>T,which changed glutamine into a stop codon,and c.766-768del,which led to amino acid alterations of p.256-256del（deletion）.The patients’parents carried the two base mutations.This study examined the clinical traits and gene identification of SCN4 offspring and parents who had the G6PC3 gene mutation.Using CRISPR/Cas9 technology,a cell line with the G6PC3 gene mutation was created as a cell model,and mutant embryos were created using the patient’s father’s sperm cells,which had a single base mutation.In order to investigate the impact and safety of cell line and embryo correction,the pathogenic genes were then corrected at the cell and embryo levels using the single base editor.These are the outcomes:（1）Using CRISPR/Cas9 and an in vitro template,a HEK293T mutant cell line was created:Using Cas9 and ss ODN,two 293^C295Tmutant cell lines were successfully created.（2）Using ABE7.10 and sg RNA to correct mutations in the 293^C295Tmutant cell line:According to the mutant base site,three different Re-sg RNAs（A13,A8 and A1）were designed,and the editing efficiency of the three Re-sg RNAs at the pathogenic site was 14.99%,19.56%and 8.97%.In the cell,the ABE7.10 editor not only edits the target site,but also the non-target site nearby.（3）Using ABE7.10 to correct pathogenic embryos:18 embryos were edited and 16 embryos were completely corrected,and the correction efficiency reached88.89%;The encoded amino acid changed from phenylalanine to either proline or leucine in 44.44%of embryos,and apparent editing processes were also found in non-target sites at the same time as the optimal correction.（4）Using deep sequencing to analyze the potential off-target sites of embryos:Using deep sequencing to detect 32 potential off-target sequences of 4sg RNA,a total of 61 sites were detected.Except for OTS11G5,the other conversion efficiencies are all below 0.5%.We have developed a novel base correction technique using human pathogenic embryos as a result of the aforementioned research.It is reliable,precise,and secure.The target sites found in the two intended sg RNAs are not in the editing window,and the editing tools are constrained by PAM sites.Additionally,some byproducts are created,changing the encoded amino acids and perhaps impacting the gene’s functionality.The basic editor built on the new Cas variant can theoretically be used to get over these restrictions and target the majority of harmful mutations.In the future,we can attempt to appropriately alter the base editor without PAM site constraints or make advantage of the currently available editor variations.This research represents the first instance of SCN4 therapy using ABE7.10.This approach offers a fresh perspective on how to treat other genetic diseases in addition to offering a novel notion for the therapy of SCN4 sickness.