Identification Of Pathogenic Point Mutations In Spinal Muscular Atrophy And Embryonic Therapy In SMA Mouse Using CRISPR/Cas9

Background and Objective Spinal Muscular Atrophy(SMA)is an autosomal recessive neurological disease that occurs more frequently in infancy.The main pathological features were selective anterior spinal horn motor neuron degeneration and the clinical manifestations were progressive symmetry limb weakness and muscle atrophy.The main pathogenic gene is the survival of the motor neuron(SMN)gene which located in the chromosome 5q13.SMN contains two highly homologous genes,SMN1 and SMN2.There are only a few single base differences between the two copies.Clinically,homozygous deletion of SMN1 gene can be found in 95% of SMA patients and the remaining 5% can present slight mutation.Due to the difference in the bases,SMN2 gene mainly encodes truncated transcripts that skip exon 7.There are two intron splicing silences,ISS-N1 and ISS+100,which play a significant role in splicing.It has been reported in relevant paper that ex8 3 'ss also plays an important role in splicing.CRISPR-cas9 system is an immune mechanism which is found in bacteria and archaea.In 1987,Japanese scholars first discovered clusters of regularly spaced short sequences near the coding region of alkaline phosphatase gene of e.coli K1,which gradually evolved into an acquired immune defense mechanism against foreign viruses and plasmids.Under the guidance of sg RNA,CRISPR/Cas9 can cut DNA.After the Double-strand DNA has been cut,the response mechanism of DNA damage repair in the cell can connect the sequences at both ends of ds DNA fracture.If there is no homologous template,the DNA sequence will be destroyed by means of non-homologous end joining(NHEJ)that may cause insertion or deletion.This study is divided into three parts.The first part is the identification of pathogenic point mutation in a SMA patient and its effect on the splicing in SMN1.In the second part,we reported a SMA pedigree with a rare genetic transmission of SMN1 mutation.In the last part,CRISPR/Cas9 gene editing technology was employed to carry out embryo intervention in SMA mouse model.Under the guidance of sg RNA,Cas9 endonuclease cut the DNA sequence of SMN2 gene 8 exon 3' splicing site,and the DNA sequence there was destroyed finally.Methods 1.Using Sanger sequencing,multiple connection dependence probe amplification(multiplex ligation-dependent probe amplification,MLPA),Polymerase chain reaction(Polymerase chain reaction,PCR),T-A cloning to analyze the SMN gene copy number and point mutation.At the same time,identificating the genotype and the rare inheritance pattern of SMN1 gene mutation in this spinal muscular atrophy pedigreethe.2.Constructed minigene was used for overexpression by transfecting p CI-minigene SMN into 293 T cells.Point mutation c.835-5 T >G p CI-minigene SMN1 was induced.At the same time,RNA was extracted to do reverse transcription PCR using p CI-minigene SMN1 and p CI-minigene SMN2 as control.RNA was extracted from the patient's peripheral blood for RT-PCR,the results were compared with normal control and a SMA patient.3.Cas9-sgrna with high cutting efficiency targeted at SMN2 8 exon 3' splicing site was constructed.Cas9 m RNA and sg RNA were injected into SMA embryos by micro-injection technology,and the changes of life span,body weight,tail length and positive turnover reflex of mice after gene editing were observed.Results 1.In the first part,SMA I patients carried one copy of SMN1 with the mutation of c.835-5 T >G.The expression level of the full-length m RNA of the SMN1 gene in the cells transfected with p CI-minigene SMN1 c.835-5 T>G was significantly decreased than that of cells transfected with p CI-minigene SMN1.Compared with patient's parents and normal control,the level of FL-SMN transcription in patient's peripheral blood was significantly reduced and the level of ?7-SMN transcription was increased.Meanwhile,the inclusion of FL-SMN transcription in patient's peripheral blood was lower than that of his parents and normal control,like that of SMA patient.Differences were statistically significant.2.In the second part,the two patients carried one copy of SMN1 with the point mutation c.844 C>T.In this family,both the mother of the proband and the father of the other patient have a copy number of 2 from SMN1 and carried the mutation c.844 C>T.Their spouses were carriers who harbored one copy of SMN1.3.In the third part,life span(more than 50 days),body weight and tail length were increased in treating severe SMA embryo(8ss-SMA I)as well as motor function.Conclusion In the first part of this study,we proved that c.835-5 T>G mutation would cause the skipping of exon 7 in most SMN1 transcripts and lead to a patient with SMA-I.In the second part of this study,the inheritance pattern of SMN1 gene mutation in the spinal muscular atrophy pedigree was rare and the c.844 C>T caused a false-positive result of PCR-RFLP,suggesting that it is necessary for us to combine the restriction enzyme digestion,MLPA,sequencing and other technologies to improve the accuracy of the genetic diagnosis.In the third part of this study,CRISPR/Cas9 gene editing technology was employed to destroy the splicing site of 3 'of SMN2 gene exon 8 in the embryos of SMA mice,with good therapeutic effect and high destruction efficiency,it will provide a new targeting site for SMA gene editing therapy in the future.