CRISPR Corrects Blood Disorder Gene

Scientists use the genome-editing technique to fix a disease-causing mutation in human cell lines.

By Kerry Grens | August 5, 2014

WIKIMEDIA, DCRJSRThe genome-editing method involving CRISPR and Cas9 has been called into duty for a wide variety of jobs, from cutting integrated HIV out of the human genome to turning off genes in primates. In a new development published today (August 5) in Genome Research, researchers have used CRISPR/Cas9 in human cell lines to rewrite a mutant gene that causes a blood disorder called β-thalassemia.

“It is an incremental step forward to use genome editing to correct disease-causing mutations,” said Paul Schmidt from Children’s Hospital Boston and Harvard Medical School who did not participate in the study. Still, he added, there are a number of hurdles that “need to be overcome before it can be used in the clinic.”

β-thalassemia is caused by a mutation in the HBB gene, resulting in a severe hemoglobin deficiency. It’s estimated to affect one in 100,000 people globally, including one in 10,000 in Europe. Patients require transfusions that can overload them with iron, a complication that also requires treatment. Some researchers are working to develop a gene therapy for the problematic gene, but so far there is no cure.

Yuet Kan of the University of California, San Francisco, and his colleagues turned to CRISPR to see if they could use it to rewrite the errant genetic code. The researchers first derived induced pluripotent stem cells (iPSCs) from the skin fibroblasts of a patient with β-thalassemia. They then designed a CRISPR/Cas9 construct to guide a corrected DNA sequence to the specified site in HBB and cut the double-stranded DNA. Homologous recombination by the cell’s own machinery then mended the DNA with the proper nucleotide sequence.

Kan’s team combined CRISPR/Cas9 with a transposon called piggyBac, which allowed the group to insert a marker of homologous recombination. “After we demonstrate certain clones had undergone homologous recombination, we can remove the piggyBac transposon by putting in an enzyme, transposase,” said Kan. In doing so, the researchers could monitor which cells respond to the treatment without having to leave in the marker.

“What’s exciting about it is there’s a combination of CRISPR technology and the piggyBac system to enable seamless gene correction” in iPSCs, said T.J. Cradick, the director of the protein engineering core facility at Georgia Tech. Cradick previously collaborated with Kan, but was not involved in this study.

Following the correction of the mutation in HBB, Kan’s team differentiated the iPSCs into hematopoietic progenitors and erythroblasts to measure their β-globin production levels. β-globin mRNA levels increased 16-fold, but the cells rarely produced adult hemoglobin. “We haven't reached the therapeutic level yet,” said Kan.

“In additional to establishing efficacies, it’s important that the cells maintain pluripotency and lack off-target effects,” Cradick told The Scientist. One of the concerns with using CRISPR is that it has the potential to snip other, unintended sites in the genome. Kan added that iPSCs are not known to transplant well, at least in mouse models, and therefore the delivery system would have to be optimized.

Although there is substantial work to be done to determine whether genome editing can offer a cure for β-thalassemia, Kan said, correcting the faulty gene shows that “here there is a path.”

F. Xie et al., “Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac,” Genome Research, doi:10.1101/gr.173427.114, 2014.

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August 11, 2014


Excerpts from: Detection of hemoglobinopathies and thalassemias using automated separation systems

1) "The hemoglobinopathies, or Hb variants, are attributable to amino acid substitution(s) in either globin chain. Currently, 1,179 total hemoglobin variants have been characterized.2"

2)"Countries with the highest incidence of diabetes also tend to have high incidence of Hb variants in the population.14"

Correlations (don't say it*) between amino acid substitutions that appear to differentiate the cell types of all cells in all individuals of all different species suggest the amino acid substitutions that differentiate cell types with hemoglobin variants and those that somehow contribute to diabetes also are nutrient-dependent. 

However, the amino acid substitutions are not likely to become fixed in the absence of their effect on DNA stability in the organized genomes of species with circulatory systems that also produce pheromones, which control the physiology of reproduction in species from microbes to man. 

For examples of other correlations in species with and without circulatory systems see: Nutrient-dependent/pheromone-controlled adaptive evolution: a model.

Is there a model of mutation-caused cell type differentiation via amino acid substitutions in species from yeasts to mammals? If not, my model may be the only model of biophysically-constrained biologically-based  cause and effect that can be compared to correlations provided in the context of theories about how mutations and natural selection somehow led to the evolution of biodiversity.


* Don't say that correlations are not causation unless you first attest to the fact that you have never thought in terms of Mutation-driven evolution without recognizing your unstated belief that correlations must be causation. If not, the theories about mutaitions are not biologically plausible theories. 

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