BATCH-GE : batch analysis of next-generation sequencing data for genome editing assessment

Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data. Therefore, we designed BATCH-GE, an easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data, available from https://github.com/WouterSteyaert/BATCH-GE.git. BATCH-GE detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel. Furthermore, this new tool provides flexibility by allowing the user to adapt a number of input variables. The performance of BATCH-GE was evaluated in two genome editing experiments, aiming to generate knock-out and knock-in zebrafish mutants. This tool will not only contribute to the evaluation of CRISPR/Cas9-based experiments, but will be of use in any genome editing experiment and has the ability to analyze data from every organism with a sequenced genome

CRISPR/Cas9‐mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse

The X‐linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas‐mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies

Cytosine deaminase base editing to restore COL7A1 in dystrophic epidermolysis bullosa human:murine skin model

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating blistering skin disorder caused by loss-of-function mutations in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils (AFs) at the dermal-epidermal junction (DEJ). Although conventional gene therapy approaches through viral vectors have been tested in pre-clinical and clinical trials, they are limited by transgene size constraints and only support unregulated gene expression. Genome editing could potentially overcome some of these limitations, and CRISPR/Cas9 has already been applied in research studies to restore COL7A1 expression. Delivery of suitable repair templates for repair of DNA cleaved by Cas9 is still major challenge, and alternative base editing strategies may offer corrective solutions for certain mutations. We demonstrate highly targeted and efficient cytidine deamination and molecular correction of a defined RDEB mutation (c.425A>G) leading to restoration of full-length C7 protein expression in primary human fibroblasts and iPSCs. C7 basement membrane expression and skin architecture were restored with de novo AFs identified by electron microscopy in base edited human RDEB grafts recovered from immunodeficient mice. The results demonstrate the potential and promise of emerging base editing technologies in tackling inherited disorders with well-defined single nucleotide mutations

Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

BACKGROUND: Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). RESULTS: We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. CONCLUSIONS: These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

Background: Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pig-to-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs. Results: We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets. Conclusions: We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation

Ride the Tide: Observing CRISPR/Cas9 genome editing by the numbers

Targeted genome editing has become a powerful genetic tool for modification of DNA sequences in their natural chromosomal context. CRISPR RNA-guided nucleases have recently emerged as an efficient targeted editing tool for multiple organisms. Hereby a double strand break is introduced at a targeted DNA site. During DNA repair genomic alterations are introduced which can change the function of the DNA code. However, our understanding of how CRISPR works is incomplete and it is still hard to predict the CRISPR activity at the precise target sites. The highly ordered structure of the eukaryotic genome may play a role in this. The organization of the genome is controlled by dynamic changes of DNA methylation, histone modification, histone variant incorporation and nucleosome remodelling. The influence of nuclear organization and chromatin structure on transcription is reasonably well known, but we are just beginning to understand its effect on genome editing by CRISP

In silico design and analysis of targeted genome editing with CRISPR

CRISPR/Cas systems have become a tool of choice for targeted genome engineering in recent years. Scientists around the world want to accelerate their research with the use of CRISPR/Cas systems, but are being slowed down by the need to understand the technology and computational steps needed for design and analysis. However, bioinformatics tools for the design and analysis of CRISPR experiments are being created to aid those scientists. For the design of CRISPR targeted genome editing experiments, CHOPCHOP has become one of the most cited and most used tools. After the initial publication of CHOPCHOP, our understanding of the CRISPR system underwent a scientific evolution. I therefore updated CHOPCHOP to accommodate the latest discoveries, such as designs for nickase and isoform targeting, machine learning algorithms for efficiency scoring and repair profile prediction, in addition to many others. On the other spectrum of genome engineering with CRISPR, there is a need for analysis of the data and validation of mutants. For the analysis of the CRISPR targeted genome editing experiments, I have created ampliCan, an R package that with the use of ‘editing aware’ alignment and automated normalization, performs precise estimation of editing efficiencies for thousands of CRISPR experiments. I have benchmarked ampliCan to display its strengths at handling a variety of editing indels, filtering out contaminant reads and performing HDR editing estimates. Both of these tools were developed with the idea that biologists without a deep understanding of CRISPR should be able to use them, and at the same time seasoned experts can adjust the settings for their purposes. I hope that these tools will facilitate adaptation of CRISPR systems for targeted genome editing and indirectly allow for great discoveries in the future

Genome editing reveals a role for OCT4 in human embryogenesis.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.DW was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. NK was supported by the University of Oxford Clarendon Fund. AB was supported by a British Heart Foundation PhD Studentship (FS/11/77/39327). LV was supported by core grant funding from the Wellcome Trust and Medical Research Council (PSAG028). J-SK was supported by the Institute for Basic Science (IBS-R021-D1). Work in the KKN and JMAT labs was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust (FC001120 and FC001193)

Applications of Designed Nucleases in Various Organisms

학위논문(박사)--서울대학교 대학원 :자연과학대학 화학부,2020. 2. 이형호.According to a development of life technology for industry and medical science, it became clear that genome editing tools will be arisen in the future. Moreover, it is necessary for understanding and finding a way of application in designed nucleases. In agreement with iAccording to a development of life technology for industry and medical science, it became clear that genome editing tools will be arisen in the future. Moreover, it is necessary for understanding and finding a way of application in designed nucleases. In agreement with its need, we can improve living resources and healthy life by utilizing designed nucleases. I have been studying genome editing for an application of various organisms using designed nucleases such as ZFN, TALEN and CRISPR. At the first study, I had modified CMAH gene in pig genome and using its knockout(KO) cells for SCNT. The CMAH gene is related to immune rejection in xenotransplantation. We had aimed to produce null CMAH organ donor pigs using by ZFN and SCNT. We improved efficiency of CMAH gene KO cells by aid of MACS and FACS surrogate reporter systems. Finally we could generate CMAH KO pig blastocysts. In the second study, we utilized TALEN for pig genome editing. In this study we had generated CMAH and GGTA1 gene KO cell lines in immortalized pig fibroblast. We could confirm a development of blastocyst using immortalized CMAH KO cell by SCNT. In the third study, we observed NR gene mutation by delivering Cas9 RNP into the protoplast of petunia. This results could suggest a possibility for gene editing in petunia by Cas9 RNP. At the last study, we showed overcoming premature termination codons (PTCs) which causes genetic defeat in a human genetic diseases. We named this method as for CRISPR-pass and proved its possibility in XPC gene patient-derived fibroblast. In a summary we tried to do gene editing in a various organisms with ZFN, TALEN and CRISPR. At the same time we also tried to help for understanding of designed nuclease and suggested a way of its applications.ts need, we can improve living resources and healthy life by utilizing designed nucleases. I have been studying genome editing for an application of various organisms using designed nucleases such as ZFN, TALEN and CRISPR. At the first study, I had modified CMAH gene in pig genome and using its knockout(KO) cells for SCNT. The CMAH gene is related to immune rejection in xenotransplantation. We had aimed to produce null CMAH organ donor pigs using by ZFN and SCNT. We improved efficiency of CMAH gene KO cells by aid of MACS and FACS surrogate reporter systems. Finally we could generate CMAH KO pig blastocysts. In the second study, we utilized TALEN for pig genome editing. In this study we had generated CMAH and GGTA1 gene KO cell lines in immortalized pig fibroblast. We could confirm a development of blastocyst using immortalized CMAH KO cell by SCNT. In the third study, we observed NR gene mutation by delivering Cas9 RNP into the protoplast of petunia. This results could suggest a possibility for gene editing in petunia by Cas9 RNP. At the last study, we showed overcoming premature termination codons (PTCs) which causes genetic defeat in a human genetic diseases. We named this method as for CRISPR-pass and proved its possibility in XPC gene patient-derived fibroblast. In a summary we tried to do gene editing in a various organisms with ZFN, TALEN and CRISPR. At the same time we also tried to help for understanding of designed nuclease and suggested a way of its applications.생명과학의 발달이 새로운 산업 동력과 미래의 대체의학으로 떠오름에 따라서 유전자 가위를 통한 생명과학 발달 가능성이 더더욱 명확해 지고 있으며, 또한 유전체에 대한 이해를 바탕으로, 이를 활용하기 위한 방법으로써 유전자 가위의 올바른 활용과 이해가 절실해지고 있다. 이에 따라 다양한 유전자 가위를 다양한 생물체의 유전체 교정에 활용함으로써, 동×식물자원의 유전형질개량을 통한 식량 및 생물자원의 증진과 나아가서는 인간 및 생물자원의 질병치료를 통한 건강한 삶을 만들어 갈 수 있을 것이다. 이러한 목표를 추구하기 위하여 ZFN, TALEN, CRISPR 과 같은 유전자 가위를 다양한 생물체에서 활용해보고자 하였다. 첫번째 연구로써는 ZFN 과 돼지 귀의 피부세포를 이용하여 유전자 교정을 통해 CMAH 유전자가 녹아웃 (Knockout)된 세포를 얻어 돼지의 난자에 체세포 핵 치환 (SCNT)을 통해 CMAH 유전자가 녹아웃 된 돼지를 생산하고자 시도하였다. CMAH 유전자는 이종장기이식에 있어서 면역거부반응을 일으키는 유전자로써 녹아웃을 통해 이종장기이식 시에 면역거부반응을 제거하고자 목표하였다. 녹아웃 세포의 확보 효율을 높이기 위해 FACS 리포터와 MACS 리포터 시스템을 활용하였으며, 이를 통해 CMAH 유전자가 녹아웃된 배아를 확인할 수 있었다. 두번째 연구에서는 TALEN을 이용하여 돼지의 CMAH와 GGTA1유전자를 녹아웃하고자 시도하였으며, 이때에는 돼지의 유전자 교정을 용이하게 하기 위해 돼지 귀의 피부세포를 불멸화 (immortalization)하여 다양한 녹아웃 세포주를 확보하고, 이를 통해 SCNT 후 배아로의 발달 과정을 확인해 볼 수 있었다. 세번째 연구에서는 CRISPR-Cas9 단백질을 페투니아의 원형질체에 전달함으로써 NR gene 이 녹아웃되는 효율을 확인하고자 목표하였으며, Cas9 RNP 전달을 통해 효율적으로 NR gene 이 녹아웃되는 것을 확인하고, 이를 통해 페투니아의 다른 유전자를 녹아웃할 수 있는 가능성을 확인하였다. 마지막으로는 CRISPR-Cas9 에 아데닌 탈아미노효소 (adenine deaminase)를 연결한 ABE 를 이용하여 미성숙 종결코돈(premature termination codon)으로 인한 질병을 치료할 수 있는 방법을 제시하고자 하였다. ClinVar 전산망에 있는 유전질활 중 미성숙 종결코돈으로 인한 비율을 확인 후, 이를 극복하기 위해서 CRISPR-pass 방법을 제시하였고, 그 치료 가능성을 XPC gene에 돌연변이가 생긴 환자의 세포에서 확인할 수 있었다. 이와 같이 ZFN, TALEN, CRISPR 뿐만 아니라 ABE와 같은 다양한 유전자 가위를 활용하여 동물과 식물 그리고 인간의 유전자 교정을 시도하여, 유전자 가위의 활용과 그 활용방안에 대한 이해를 돕고자 하였다.PART 1. Applications of designed nucleases: Zinc Finger Nuclease (ZFN), TAL Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 1 Ⅰ. Introduction 2 Ⅱ. Materials and Methods 8 1. Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System 8 a. ZFNs and surrogate reporters 8 b. Preparation of cells and culture conditions 8 c. Plasmid DNAs transfection 8 d. Analysis of mutations 9 e. Production and culture of cloned porcine embryos 9 f. Statistical analysis 10 2. Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases 11 a. Chemicals . 11 b. Primary cell culture and maintenance. 11 c. Immortalization 11 d. Doubling time 12 e. Cell size 12 f. PCR 12 g. Sequencing 12 h. Karyotyping 13 i. Single cell colony formation 13 j. Gene expression 14 k. Telomerase activity test 14 l. Nuclear transfer 14 m. CMAH knockout using TALEN and magnetic separation 15 n. T7E1 assay 15 o. Fluorescent PCR 15 p. Fluorescence-activated cell sorting 16 q. Statistical analysis 16 3. Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins 16 a. Petunia protoplast preparation 16 b. Recombinant Cas9 Protein and Guide RNA design 17 c. Transfection 17 d. Genomic DNA extraction and T7 endonuclease 1 (T7E1) assay 18 e. Targeted deep sequencing 18 Ⅲ. Results 20 1. Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System 18 a. Enrichment system for cells containing ZFN-mediated mutations 18 b. Mutations in cloned blastocysts derived from ZFN-treated cells 24 2. Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases 27 a. Generation of porcine immortalized cell and analysis of immortalized cells properties 27 b. Preimplantation development of cloned embryos derived from immortalized cells 32 c. CMAH knockout and SCNT 32 d. GGTA1 knockout 43 3. Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins 51 a. Efficient protoplast system enhances Cas9 transfection in P. hybrida 51 b. Targeted mutagenesis of NR gene in Petunia protoplast system using direct delivery of RGEN RNPs 52 c. Detection and estimation of Cas9/sgRNA mediated Petunia NR gene mutations. 60 Ⅳ. Discussion 68 PART 2. Application of designed nuclease: Adenine base editor (ABE) 69 Ⅰ. Introduction 70 Ⅱ. Materials and Methods 72 1. General Methods and Cloning 72 2. ClinVar Database Analysis 72 3. Cell Culture and Transfection 73 4. EGFP-PTC-KI Cell Lines 74 5. Flow Cytometry 74 6. Targeted Deep Sequencing 74 7. Treatment with Ataluren and Gentamicin 75 8. Western Blotting 75 9. Functional Assessment 75 10. Statistics 76 11. Data Availability 76 Ⅲ. Results 77 1. In Silico Investigation of Applicable Targets for CRISPR-Pass in the ClinVar Database 77 2. Construction of Six KI HeLa Cell Lines Carrying Various Types of PTCs in EGFP Gene 79 3. CRISPR-Pass Rescues the Function of the EGFP Gene in Six KI HeLa Cell Lines 81 4. CRISPR-Pass Rescues the Function of the XPC Gene in Patient-Derived Fibroblasts 91 Ⅳ. Discussion 104 References 106 Abstract in Korean 119Docto