In vitro correction of hbb v6g mutation in lymphocyte cells of patients with sickle cell anemia using genome-editing crispr / cas9 technique
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Tarih
2018
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Orak hücre hastalığı OHA kırmızı kan hücrelerinde oksijen taşıyan bir protein olan hemoglobinin anormal oluşumu ile karakterize bir kalıtsal kan hastalığıdır. Orak hücreli anemi akciğerden vücuttaki hücrelere oksije taşıyan hemoglobinin mutant formu tarafından meydana getirilmektedir. Hemoglobin farklı genler tarafından kodlanan a-globin ve b-globin olamak üzere iki polipeptiden oluşmuştur. Herbir fonksiyonel hemoglobin molekülü iki a-globin ve iki b-globini içerir. Orak hücreli anemide b-globini kodlayan gendeki bir mutasyon proteindeki 146 aminoasitten birinde bir aminoasit yer değişikliğine yolaçmaktadır. Bakıldığında orak hücreli anemide ki mutasyon bir DNA nükleotinde değişikliği olup mRNA'da kodon 6 daki GAG dizisini GUG şeklinde dönüştürmekte, buda b-globinde glutamik asit yerine valin gelmesinde yolaçmaktadır. Proteindeki diğer aminoasitler bu mutasyondan etkilenmemektedir. Bu ciddi hastalık dünya çapında milyonlarca çocuğu etkilemektedir. Genom düzenleme tekniklerindeki son gelişmeler programlanabilir nükleazlar kullanılarak istenen herhangi bir DNA dizisini değiştirmek mümkün hale getirmiştir. Bu yeni nesil genom değiştirici araçlar özellikle orak hücre hastalık OHA gibi hastalıkların tedavesine yönelik terapötik uygulamalar için ideal adaylardır. OHA için destekleyici tedavi stratejilerinin geliştirilmesinde önemli bir başarı elde edilmiştir, ama ne yazık ki hala uzun vadeli evrensel tedavi eksikliği vardır. Günümüzde mevcut küratif tedavi sağlıklı vericilerden allojenik kök hücre transplantasyonu dayanmaktadır; Bununla birlikte, bu tedavi, sadece sınırlı sayıda hastalara uygulanabilmektedir. Genome editing teknolojilerinde son birkaç yıl içerisinde önemli gelişmeler sağlandı. Günümüzde yaygın olarak kullanılan genom editing teknoljileri ZFN (Zinc Finger Nucleases), TALEN (Transc ription Activator-Like Effector Nucleases) ve CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) teknolojileridir. ZFN and TALEN genomik bölgeyi hedfelemek için DNA binding domaine eklenen endonükleazlardan oluşturulurken, CRISPR`da bu guide RNA ve Cas9 aracığıyla gerçekleşmektedir. Bu endonükleazlar aracılığıyla oluşturulan çift DNA zincir kırıkları homolog direkt tamir veya homolog olmayan uç birleştirmeleri yoluyla tamir edilmekte ve buda insersiyon veya delesyonlarla sonuçlanmaktadır. Bu teknolojiler hedef genom bölgelerinin düzeltilmesinde hücrenin normal DNA tamir mekanizmlarını kullanmakta ve değişik genetik hastalıklarda genomun manupülasyonu amacıyla uygulanmaktadır. Bu genom editing araçları ayrıca gen fonksiyon analizi, yeni tedavi hedeflerinin bulunması ve yeni yolakların araştırılmasında kullanılabilmektedir. Projemizde CRISPR/Cas9 tekniği kullanılarak sickle cell anemia neden olan V6 G mutasyonunu yok etmeyi amaçlandı. Projemizde V6G mutasyonunu içeren orak hücrel anemili vakalardan elde edilen lenfosit hücreleri kullandık. Bunun için öncelikle hedeflediğimiz bölgeye özgü rehber RNA'yı dizayn ettik. Bu rehber RNA sekansını (gRNA) vektöre aktardıktan sonra vektörü E. coli bakteri hücresinde çoğaltik. Bakteri kültüründen plazmid izolasyonundan sonra, vektörü Polyethylenimine (PEI) transfeksiyon yoluyla lenfosit hücrelerine transfekte ettik. Hücre kültürü, sterile koşullarda bir veya iki hafta daha devam edildi. Ardından DNA izolasyonunu takiben Knockout'u doğrulamak için real-time PCR analizi yapıldı.
Sickle-cell disease (SCD), also known as sickle-cell anemia, is a hereditary blood disorder characterized by the presence of abnormal hemoglobin, the oxygen-carrying protein found in red blood cells. Sickle-cell anemia is caused by a mutant form of hemoglobin, the protein that transports oxygen from the lungs to cells in the body. Hemoglobin is a composite molecule made up of two different polypeptides, a-globin and b globin, each encoded by a different gene. Each functional hemoglobin molecule contains two a-globin and two b globin chains. In sickle-cell anemia, a mutation in the gene encoding b-globin causes an amino acid substitution in 1 of the 146 amino acids in the protein. Notice that the mutation in sickle-cell anemia consists of a change in one DNA nucleotide, which leads to a change in codon 6 in mRNA from GAG to GUG, which in turn changes amino acid number 6 in b-globin from glutamic acid to valine. The other 145 amino acids in the protein are not changed by this mutation. This devastating hematologic disease affects millions of children worldwide. Recent advances in genome-editing techniques have made it possible to modify any desired DNA sequence by employing programmable nucleases. These next-generation genome-modifying tools are the ideal candidates for therapeutic applications, especially for the treatment of genetic disorders like sickle cell disease (SCD). Substantial success has been achieved in the development of supportive therapeutic strategies for SCD, but unfortunately there is still a lack of long-term universal cure. The only existing curative treatment is based on allogeneic stem cell transplantation from healthy donors; however, this treatment is applicable to a limited number of patients only. Genome editing technologies have improved significantly in the last few years. Currently, The commonly applied genome editing technologies are Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). ZFNs and TALENs consist of endonucleases joined to a DNA-binding domain while the CRISPR utilizes guide RNAs and Cas9 to targeted the genomic location. The double-strand breaks produced by these endonucleases are repaired in the cells either by homology directed repair (HDR) or non-homologous end joining, resulting in insertions/deletions. These technologies utilize normal DNA repair mechanisms of the cell for correctionof the targeted genome site and have been applied to manipulate the genome in different genetic disorder. These genome editing tools can be also utilized to analyze gene function, to identify new therapeutic targets, and to explore new pathway. By using the CRISPR / Cas9 technique in our project, we aimed to knock out the V6G mutation that causes sickle cell anemia. In our project, we used the lymphocyte-containing the V6G mutation obtained in the sickle cell anemia. For this, we first designed the region-specific guide RNA we are targeting. After transferring this guide RNA sequence (gRNA) to the vector, we propagated the vector in the E. coli bacterial cell. After plasmid isolation from bacterial culture, we transfect the vector by Polyethylenimine (PEI) transfection methods into the lymphocyte cells. The cell culture continued for one or two more weeks in sterile conditions. DNA isolation from the cells followed by Real-time PCR analysis performed to confirm the knockout.
Sickle-cell disease (SCD), also known as sickle-cell anemia, is a hereditary blood disorder characterized by the presence of abnormal hemoglobin, the oxygen-carrying protein found in red blood cells. Sickle-cell anemia is caused by a mutant form of hemoglobin, the protein that transports oxygen from the lungs to cells in the body. Hemoglobin is a composite molecule made up of two different polypeptides, a-globin and b globin, each encoded by a different gene. Each functional hemoglobin molecule contains two a-globin and two b globin chains. In sickle-cell anemia, a mutation in the gene encoding b-globin causes an amino acid substitution in 1 of the 146 amino acids in the protein. Notice that the mutation in sickle-cell anemia consists of a change in one DNA nucleotide, which leads to a change in codon 6 in mRNA from GAG to GUG, which in turn changes amino acid number 6 in b-globin from glutamic acid to valine. The other 145 amino acids in the protein are not changed by this mutation. This devastating hematologic disease affects millions of children worldwide. Recent advances in genome-editing techniques have made it possible to modify any desired DNA sequence by employing programmable nucleases. These next-generation genome-modifying tools are the ideal candidates for therapeutic applications, especially for the treatment of genetic disorders like sickle cell disease (SCD). Substantial success has been achieved in the development of supportive therapeutic strategies for SCD, but unfortunately there is still a lack of long-term universal cure. The only existing curative treatment is based on allogeneic stem cell transplantation from healthy donors; however, this treatment is applicable to a limited number of patients only. Genome editing technologies have improved significantly in the last few years. Currently, The commonly applied genome editing technologies are Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). ZFNs and TALENs consist of endonucleases joined to a DNA-binding domain while the CRISPR utilizes guide RNAs and Cas9 to targeted the genomic location. The double-strand breaks produced by these endonucleases are repaired in the cells either by homology directed repair (HDR) or non-homologous end joining, resulting in insertions/deletions. These technologies utilize normal DNA repair mechanisms of the cell for correctionof the targeted genome site and have been applied to manipulate the genome in different genetic disorder. These genome editing tools can be also utilized to analyze gene function, to identify new therapeutic targets, and to explore new pathway. By using the CRISPR / Cas9 technique in our project, we aimed to knock out the V6G mutation that causes sickle cell anemia. In our project, we used the lymphocyte-containing the V6G mutation obtained in the sickle cell anemia. For this, we first designed the region-specific guide RNA we are targeting. After transferring this guide RNA sequence (gRNA) to the vector, we propagated the vector in the E. coli bacterial cell. After plasmid isolation from bacterial culture, we transfect the vector by Polyethylenimine (PEI) transfection methods into the lymphocyte cells. The cell culture continued for one or two more weeks in sterile conditions. DNA isolation from the cells followed by Real-time PCR analysis performed to confirm the knockout.
Açıklama
Anahtar Kelimeler
Sickle cell anemia, Genome editing, Orak hücreli anemi, CRISPR/Cas9, Kan hastalıkları, Hematologic diseases
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Zainalbden, H. A. Z. (2018). In vitro correction of hbb v6g mutation in lymphocyte cells of patients with sickle cell anemia using genome-editing crispr / cas9 technique. Selçuk Üniversitesi, Yayımlanmış yüksek lisans tezi, Konya.
