The Optimization Of CRISPR/Cas9 And Its Application In Human Stem Cells

The discovery and development of gene editing technology based on CRISPR/Cas9 system has provided a new strategy for precision medicine and a novel platform for treating human diseases,especially rare genetic diseases.The improvement of gene-editing efficiency in stem cells by minimizing and evaluating off-target effects is the prerequisite and key of CRISPR/Cas9 system to clinical applications.In this paper,by means of spontaneous non-homologous end joining(NHEJ)or homologous directed repair(HDR)after DNA double-strand breaks(DSBs),one or more CRISPR/Cas9 system targeting specific gene sequence were introduced into human cell lines or stem cells.The DNA double-strand breaks were triggered,and thus induced the insertion,modifications,and deletions of gene sequences which improved the efficiency and accuracy of targeted editing.By taking advantage of the preferential recognition to DNA double strand breaks by Integration-defective Lentiviral vectors(IDLVs),we evaluated the off-target effects of CRISPR/Cas9 system and optimized the genetic modification efficiency in stem cells.Chapter 1:A new approach for unbiased detection of off-target cleavage by CRISPR/Cas9In recent years,CRISPR/Cas9 system has been widely used in fundamental research of animals and plants,and it is highly expected to be employed in the future clinical treatment.However,the off-target effects remains to interfere its application,whereas the existing network analysis tools and in-vitro validation method aiming off-target sites can not offer detailed off-target conditions within cells.In order to utilize the CRISPR/Cas9 system in a secure and accurate way,it is important to comprehensively screen the off-target sites within cells after genetic editing.It has been reported that,the genome of linear double-stranded IDLV can identify and integrate into double strand breaks indeuced by ZFNs through non-homologous end joining repair.We innovatively applied this genome-wide detection system to screen the off-target sites created by specific CRISPR/Cas9.We designed 3 gRNAs and one pair of TALEN as comparison for Wiskott-Aldrich Sydrome(WAS)and Tyrosine Aminotransferase(TAT),which are two disease-causing mutations.The HEK293T cells were firstly transfected with CRISPR/Cas9 or TALEN,and then transduced with IDLV containing puromycin selective marker on the second day.The puromycin positive clones were collected and the whole genome DNA were extracted.With the help of nonrestrictive(nr)linear amplification-mediated PCR(LAM-PCR)and deep sequencing,we were able to locate and analyze the Clustered IDLV Integration Sites(CLISs).We found that the off-target rate of gRNA mediated by CRISPR/Cas9 system was totally different between each gRNAs.And when gRNA possessed one to two more or one to two less bases than the DNA of recognition site,a bulge was formed during gRNA marrying DNA and double-strand breaks were still be able to be induced.This kind of off-target sites were beyond the current network algorithms.This study confirmed and determined the off-target effects of CRISPR/Cas9 system.The method was sensitive enough to detect the off-target effects with cutting efficiency in no less than 1%.Although not all the off-target sites can be detected,this method was able to unbiased screen the off-target sites at genome-wide level.As a biological analytical method,it offered a new idea and evaluation platform to improve CRISPR/Cas9 system.Chapter 2:Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cellsPluripotent Stem Cells(PSCs),such as Embryonic Stem Cells(ESCs)and induced Pluripotent Stem Cells(iPSCs),act as a tool in the studies of developmental biology and pathology mechanism.The PSCs carrying disease genes are able to differentiate into cells or tissues with the mutation,which are appropriate objectives for high-throughput toxicology/efficacy screening.This PSCs based in-vitro system is expected to fill in the gap between animal models and clinical trials and reduce the cost during drug development.Previous studies reported that the accurate gene editing conducted in human PSCs is not efficient,and we aimed to improve this.Since CRISPR/Cas system is capable of triggering double-strand breaks at target gene,it will induce accurate gene editing in the manner of homologous recombination if sufficient donor template were available.Thus,we designed 2 gRNAs target the site with high mutation rate in the intron 6 of WAS gene.With the help of a donor template delivered by IDLV to carry on accurate genetic modification in hESCs.We found the rate of homologous recombination exceeded 50%.As the main protein to promote intracellular homologous recombination repair,Lens Epithelium-Derived Growth Factor(LEDGF/p75)was confirmed to interact with Integrase(IN)of IDLV and bring the template sequence to the targeted site.This study optimized the gene editing efficiency in human PSCs,and firstly verified the function of LEDGF/p75 protein in homologous recombination repair when IDLV was used to carry the donor template.Chapter 3:CRISPR/Cas9 mediated targeted DNA sequence deletoin in the treatment of ?-hemoglobinopathies?-thalassemia and Sickle cell disease(SCD)are two kinds of single-gene hereditary diseases which have been thoroughly studied.The subunit mutations of ?-globin led abnormal structures of Adult Hemoglobin(HbA),thereby generated the malfunctioned hemoglobin.The severity of the disease has a close relationship with the expression level of Fetal Hemoglobin(HbF)in patient,thus the symptoms can be alleviated by increasing the content of HbF within hemoglobin.Previous studies have shown that a 10 kb fragment in the second intron of BCL11A gene can specifically enhance the expression of BCL11A gene in normal erythroid development,thus inhibiting the expression of y-globin.Therefore,by deleting this specific enhancer,the expression of BCL11A gene was suppressed,which increased the amount of HbF.This method can be used as a new strategy for alleviating ?-hemoglobinopathies.To delete the specific enhancer region on BCL11A gene,we designed a pair of gRNA targeting both sides of the region.We introduced CRISPR/Cas9 system containing two gRNAs into Human Umbilical Cord Blood Derived Erythoid Progenitor-2 to build cell lines with homozygous and heterozygous deletion.Then we examined the expressions of BCL11A and y-globin.In addition,we apply this strategy in human CD34+hematopoietic stem cells.We discovered the deletion efficiency of large fragment in hematopoietic stem cells was far below that in cell lines.Comparing to control group,the proportion of y-globin in?-like globin were increased from 1%to 45%in homozygously enhancer deficient cell lines,and 1%to 20%in heterozygotes.And transfecting large plasmid in the manner of electroporation caused lots of cell death.This study verified the negative correlationship between y-globin and BCL11A gene.Modifying the regulatory factors of disease gene,rather than directly altering genetic site,shows a great advantage in the clinical applications.