SgRNA Optimization And CRISPR/Cas9-mediated Treatment On Epidermolytic Palmoplantar Keratoderma-like Mouse

ObjectivesGenodermatoses,or hereditary skin disorders,constitute a group of rare heterogeneous diseases,bearing congenital,familial,and lifelong characteristics as other genetic conditions,which may lead to the suffering of life unbalance even life threatening.Among the hereditary palmoplantar keratodermas（PPKs）,epidermolytic palmoplantar keratoderma（EPPK,OMIM:#144200）inherited in an autosomal dominant pattern with a worldwide incidence of 2.2 to 4.4 per 100 000 live births,is the most common subtype.The symptoms of EPPK usually onset in few years after born and last for lifetime,which have a severely negative impact on the patients and their families.So far,there is no radical treatment for EPPK.Therefore,it is urgent to develop a novel therapy.Gene therapy is the best therapeutic option for monogenic diseases,and CRISPR/Cas9,which was awarded Nobel Prize for Chemistry in 2020,is now widely acknowledged as the most effective genome editing tool.Design of small guide RNA（sgRNA）plays the critical role in determining the working efficiency of CRISPR/Cas9.Majority of hereditary diseases,including EPPK,were diagnosed after born.If the current researches were focused on gene editing the fertilized egg,these efforts would be unhelpful to treat the genetic disorders.Here,our study aims to investigate whether CRISPR/Cas9 is a potentially powerful therapeutic option for humanized EPPK-like mouse by injecting CRISPR/Cas9 into lesions locally,and discuss the sgRNA optimization rules.Methods and Results（1）Based on the Krt9/c.434delAinsGGCT humanized EPPK-like mouse we established previously,multiple sgRNAs were designed to target the Krt9/c.434delAinsGGCT mutation（MUT allele）.By the principle that“The closer sgRNA and target site are,the more efficient CRSPR/Cas9 will be”,a sgRNA cutting on MUT allele was selected.And based on the principle that“PAM on the mutations will improve the efficiency of CRISPR/Cas9”,a novel PAM site on MUT allele was generated to design the sgRNA.Thus,two sgRNAs which ranked 4^th and 7^th on Zhang Lab website（crispr.mit.edu）were determined.（2）We constructed two cell lines stably co-expressing Krt9/c.434 wide type（WT）allele-luciferase and MUT allele-luciferase,respectively.Some reports had suggested that the effects of scr7 and ss ODN on homology directed repair（HDR）and non-homologous end joining（NHEJ）were remarkable.However,these effects were not detected after statistical analysis in our experiments using cells expressing MUT allele.Thus,scr7 and ss ODN were abandoned in the following procedures.By using the cells co-expressing MUT allele and luciferase,the luciferase down-regulation expression（cutting potency on target site）caused by sgRNA1-Cas9-LV was 0.49 times higher than that of sgRNA2-Cas9-LV.Overall,sgRNA1-Cas9-LV had higher cutting efficiency than sgRNA2-Cas9-LV by using cells expressing MUT allele.（3）We constructed a cell line co-expressing WT allele-m Cherry and MUT allele-enhanced green fluorescent protein（eGFP）,i.e.heterozygous for Krt9（Hez cells）.The allele-specificity and potency of sgRNA-1 and sgRNA-2 were assessed in vitro mimicking the EPPK heterozygotes.After the transduction of sgRNA1-Cas9-LV,the potent reductions of 63.2%and 22.8%for total eGFP fluorescence（expression of MUT allele）and m Cherry fluorescence（expression of WT allele）were observed in Hez cells,compared with lesser reductions of 32.1%and 7.0%measured in the cells infected with sgRNA2-Cas9-LV,respectively.FLAG expressed in cells infected with lentivirus.Using the expression levels of FLAG as references,we found 43.5%vs 21.6%reductions of K9in the cells infected with sgRNA1-Cas9-LV and sgRNA2-Cas9-LV,respectively.These data suggested that the potency of sgRNA1-Cas9-LV was higher than that of sgRNA2-Cas9-LV,and the reduction of K9 expression was more obvious.（4）In the humanized EPPK-like mouse model,the phenotype developed on the major impact-dampening footpads of the main weight-bearing paws,the fore-paws.To determine whether sg1-Cas9-LV affects the process of hyperkeratosis in vivo,12-week-old KI-Krt9 mice were treated with subcutaneous injections of sg1-Cas9-LV into the right fore-paw 3 times every 8 days.The left fore-paw of the model mice was injected with Cas9-LV as controls.After CRISPR/Cas9 management on EPPK-like mouse model,the epidermal thickness was decreased with less expansion of the epidermis and less massive hyperkeratosis.The average epidermal thickness of the right pads was 43μm,half of 86μm which was the thickness of left pads（0.01<P<0.05）,and the level of melanin was also reduced.After treatment,mice showed fewer abnormal tonofilaments and fewer cytolysis-caused vacuoles in the suprabasal layers of the epidermis.The expression level of K1,K5,K6,involucrin and filaggrin were resemble to those in WT mice instead of humanized EPPK-like mouse model,the abnormal proliferation was reduced.（5）Among humanized EPPK-like mice treated with sgRNA1-Cas9-LV,Sanger sequencing of Krt9/c.434 WT allele and top 10 potential highest-ranking genomic off-target sites was performed to assess the off-target effects.Although no off-target sequencing on WT allele were observed,DNA sequencing of the top 10 potential highest-ranking genomic showed 1.5%off-target effects.Conclusions（1）Evaluation from histological phenotype,protein expression level,locations of protein expression,microstructure and other aspects indicated the amelioration of EPPK-like phenotype of mice after the treatment of CRISPR/Cas9.Thus,clinical trials of EPPK using CRISPR/Cas9 could be considered in the further study.（2）Based on our observation,it suggested that for monogenic mutations,the sgRNA with the cut-site mutation（sgRNA1）is more efficient than that with the PAM-site mutation（sgRNA2）.This sgRNA optimization rule may be a reference for the other CRISPR/Cas9 therapies on monogenic disorders.