Study On Repair Efficiency Of Human Zygotes Carrying PAH Mutation By CRISPR/Cas9

Objective: To investigate the effect of inner cell mass isolation methods on the establishment of human embryonic stem cells using IVF discarded embryos,and to establish PGD-derived hESCs containing PAH mutation from patients.Methods: 10 discarded fertilized embryos after IVF treatment and 2 embryos containing PAH mutation after PGD treatment were collected in this study.IVF-derived embryos group A(1#-5#)and PGD-derived number 2 blastocyst were treated by laser boring combined with mechanical stress dissection.IVF-derived embryos group B(6#-10#)and PGD-derived number 4 blastocyst were treated by intact embryo culture methods.These two methods were compared by the primary clone formation rate and the capacity of passage number.The established hESCs were characterized when a steady growth of colonies were reached.Results: By comparing group A with group B,there was no difference in inner cell mass attachment,but the primary clone formation rate of group A(60%,3/5)was higher than that of group B(20%,1/5).Finally,only group A 1# were passaged successfully.Because of the overgrowth of trophoblastic cells,PGD-derived number 4 primary clone died after passage.hESC was isolated successfully from PGD-derived number 2 blastocyst,which carried PAH mutation from the paternal patient.Characterization of hESC showed that it was AKP positive,the karyotype analysis displayed intact diploid chromosomes,the stem cell markers SSEA-3,SSEA-4 and OCT-4 were expressed.hESCs also differentiated into three germ layers both in vivo and in vitro.Conclusions: Laser boring combined with mechanical stress dissection can improve the success rate of human embryonic stem cell establishment;We successfully established PGD-derived human embryonic stem cells containing PAH gene mutation.Objective: To study repair efficiency and safety of human zygotes carrying PAH pathogenic mutations using CRISPR/Cas9 technology,and to provide a preliminary research base for the treatment of phenylketonuria.Methods: Firstly,the targeting efficiency of the designed three kinds of sg RNA was verified on human embryonic stem cells carrying PAH pathogenic mutations,and the most efficient sg RNA was selected for in vitro transcription;The mature oocytes were divided into experimental group and control group,for the experimental group,the mature oocytes were injected of patient's sperm mixed with sg RNA,Cas9 protein and ss ODN,for the control group only injected the patient's sperm.Then analyze the effect of CRISPR/Cas9 effect on embryonic development,the targeting results and repair efficiency of PAH mutation by CRISPR/Cas9,and the possible safety issues of this method.Results: The targeting efficiency of hs-PAH-ngg-sg in human embryonic stem cells was the highest;The survival rate,2PN fertilization rate,cleavage rate and high-quality embryo rate of the control group were higher than those of the test group,but there was no significant difference(P>0.05).The proportion of embryos with WT/WT alleles in the experimental group(80%)was higher than that in the control group(45%),the difference was statistically significant(P<0.05).The targeting efficiency to MUT allele was 100%,in which the efficiency of HDR by ss ODN was 25%(incomplete recombination);the off-target rate was 5.56% toward WT allele,and no off-targeting was found among other possible sites.Conclusions: The targeting efficiency of PAH pathogenic mutation in human zygotes using CRISPR/Cas9 was 100%,and the efficiency of partial HDR by ss ODN was 25%. The proportion of wild-type genotypes was significantly increased,and the off-target rate of WT alleles was 5.56%.NHEJ repair resulted in random insertion or deletion mutations.The feasibility and safety of human embryo editing still need further careful study and evaluation.