CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia

2021 ◽  
Author(s):  
Haydar Frangoul ◽  
David Altshuler ◽  
M. Domenica Cappellini ◽  
Yi-Shan Chen ◽  
Jennifer Domm ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Gene Editing ◽  
Cell Disease

CRISPR gene editing for sickle cell disease: one disease, differentiated approaches

2021 ◽  
Vol 7 (7) ◽  
pp. 851-856
Author(s):  
Lisa A Michaels, MD ◽  
Bruce E Eaton, PhD
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Gene Editing ◽  
Cell Disease

CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia

2021 ◽  
Vol 76 (6) ◽  
pp. 327-329
Author(s):  
Haydar Frangoul ◽  
David Altshuler ◽  
M. Domenica Cappellini ◽  
Yi-Shan Chen ◽  
Jennifer Domm ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Gene Editing ◽  
Cell Disease

scholarly journals CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia

2020 ◽  
Author(s):  
Haydar Frangoul ◽  
David Altshuler ◽  
M. Domenica Cappellini ◽  
Yi-Shan Chen ◽  
Jennifer Domm ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Gene Editing ◽  
Cell Disease

A Highly Sensitive Assessment Tool for the Gene Editing Efficiency of Sickle Cell Disease Hemoglobin Beta Gene

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5557-5557
Author(s):  
Mandula Borjigin ◽  
Eric Brian Kmiec ◽  
Rigumula Wu
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Point Mutation ◽  
Sickle Cell ◽  
Gel Electrophoresis ◽  
Gene Editing ◽  
Cell Disease ◽  
Transcription Activator ◽  
Hematopoietic Stem ◽  
Editing Efficiency

Abstract In sickle cell disease, a single point mutation in hemoglobin β gene (HBB) results in the substitution of valine for glutamic acid at position 6 of the β globin protein sequence, causing the deformation of red blood cells into a sickle (or crescent) shape. With the development of powerful gene editing tools, scientists are initiating the correction of the point mutation of HBB gene in CD34+ hematopoietic stem cells and induced pluripotent stem cells. Although the results are very exciting, the evaluation method of the gene editing is primitive. Currently, the modification at the mutation site is identified and quantified using Restriction Fragment Length Polymorphism (RFLP), which involves PCR amplification, restriction enzyme digestion and gel electrophoresis. The accuracy of the gene editing efficiency depends heavily on the quantification of the DNA bands in the gel images, which is inherently imprecise. We have developed a novel technique to quantify the correction efficiency of HBB gene editing using a fluorescence tagging of the edited DNA sequence. This method provides excellent sensitivity and accuracy, and saves time and labor, eliminating a process of gel electrophoresis. We demonstrate the assessment of gene editing in HBB of K562 cells, in which the wild type HBB (βA gene) is converted to mutant βs using the gene editing tools (i.e. Transcription Activator-Like Effector Nucleases (TALENs) and single-stranded oligo deoxynucleotides (ssODNs)). We present limited information here due to the sensitivity of the intellectual property, but will discuss in detail the experimental procedures and data at the American Society of Hematology meeting. Disclosures No relevant conflicts of interest to declare.

Target Site Mutagenesis during Crispr/ Cas 9/Single-Stranded- Oligonucleotide Directed Gene Editing for Sickle Cell Anemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4706-4706 ◽  
Author(s):  
Dula Man ◽  
Brett Sansbury ◽  
Pawel Bialk ◽  
Kevin Bloh ◽  
E. Anders Kolb ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sanger Sequencing ◽  
Gene Editing ◽  
Globin Gene ◽  
Beta Globin ◽  
Cell Disease ◽  
Beta Globin Gene ◽  
Site Mutagenesis ◽  
Single Stranded Oligonucleotide

Abstract Introduction Sickle Cell Disease (SCD) results from a simple substitution of valine for glutamic acid at codon 6 in the β globin gene, resulting in an AàT transition in the third position. The mutation results in the production of hemoglobin HbS which differs from the normal HbAin that it tends to polymerize into long strands that deform the erythrocyte. While a variety of traditional treatment regimens or reagents, such as hydroxyurea and chronic transfusions have been used widely, these therapies are wrought with short and long term side effects that limit efficacy. There is great interest in the hypothesis that the repair of a single nucleotide is facilitated by the combined action of CRISPR/Cas 9 and single-stranded oligonucleotides (ssODNs) could prove a significant therapeutic advance for sickle cell disease. CRISPR/Cas 9 induces a site-specific double-stranded break while the single-stranded oligonucleotide provides a DNA template to improve the rate of accurate genetic correction. There is a great effort to understand off-site mutagenesis caused by editing of DNA regions remote to the target sequence. It is critical that investigators understand the frequency and types of DNA alterations induced by the gene editing reaction and affecting the region surrounding the targeted nucleotide site (herein termed on-site mutagenesis). Methods We targeted beta globin genes with a CRISPR/Cas9 system designed to cleave 2 bases to the 5'side of the targeted (A) nucleotide (the PAM site is located 2 bases to the 3' side on the complimentary strand) coupled to the electroporation of a single-stranded oligonucleotide (72-mer) designed to convert the wild-type (A) to the mutant (T) nucleotide. To evaluate the rate of on-site mutagenesis among individual alleles, we clonally expanded populations of edited K562 cells. We hypothesize that the evaluation of individual clones will permit a clear identification of intended and un-intended on-site DNA alteration. Allelic heterogeneity is analyzed using Tracking of Indels by DEcompositon (TIDE) methodology combined with Sanger sequencing. Results We isolated 26 clonal lines for continued expansion after evidence of CRISPR/Cas 9 plasmid uptake and activity. Sanger sequencing with TIDE analysis revealed that the DNA sequence surrounding the targeted base is altered significantly as a result of the CRISPR/Cas9 gene editing process. Twenty three percent of the clones contain at least one corrected allele but one hundred percent of the clones exhibited mutagenicity of the DNA sequence surrounding the targeted base. All clones analyzed displayed varying degrees of sequence alteration (Figure 1). Interestingly, one clone contained a DNA insertion homologous to a region of the delta globin gene, suggesting that gene editing of the beta globin gene may have been repaired, in part, by delta globin DNA. The sequence of the delta globin locus was not changed. Conclusions Taken together, our data suggest that combinatorial approaches to beta globin gene editing using CRISPR/Cas 9 and single-stranded oligonucleotides induce significant onsite mutagenesis and potential genetic swapping between related members of the same gene family. These observations provide insight into the type of molecular activity that accompanies combinatorial gene editing, particularly surrounding the target site. This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM109021 Disclosures No relevant conflicts of interest to declare.

CRISPR cuts disease course short in blood disorders

2021 ◽  
Vol 13 (575) ◽  
pp. eabg1756
Author(s):  
Bence György
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Gene Editing ◽  
Disease Course ◽  
Cell Disease ◽  
Blood Disorders

Gene editing for sickle cell disease and β-thalassemia results in potential curative response.

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