CRISPR/Cas gene editing in lab-grown mini organs: The intersection of two new applications to study hard-to-tackle diseases
CRISPR/Cas gene editing technology has taken the world by storm, while lab-grown mini-organs, also known as organoids, have steadily grown in their experimental potential to model their respective organs. Recently, a few research groups have started to combine both techniques, such as the team led by Dr. Deyou Zheng at the Albert Einstein College of Medicine in the United States, who used CRISPR/Cas to study genetic implications of autism in brain organoids. Combining these techniques opens up new possibilities to study diseases and uncover new therapeutic targets.
Using CRISPR/Cas to study genetic disorders
CRISPR/Cas, or clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas), is the latest advance in gene editing techniques. CRISPR/Cas can be used to efficiently generate mutations in cultured cell lines and in animal models to mirror genetic mutations in human diseases.
Using brain organoids to study autism
3D culturing of human brain organoids is a promising tool to study neuropsychiatric disorders such as autism and schizophrenia. Neuropsychiatric disorders are said to be unique to humans, and are the result of a multitude of genetic and environmental factors, making it difficult to study such diseases using animal models. Additionally, Dr. Zheng told CamBioScience the advantages of culturing brain organoids, highlighting that “human brain functions cannot be modelled by monolayer cells due to the lack of 3D interactions between cell types and brain sub-regions.”
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Combining CRISPR/Cas with brain organoid research
Over half of autism cases can be attributed to genetic mutations, although only 1% of autism cases are caused by a single mutation, termed monogenic autism. However, studying monogenic forms of autism can provide great insight into the disorder, “because even though a single gene is affected, similar pathways may be affected in other forms of autism,” said Teresa Tavassoli, PhD, a lecturer at the University of Reading in the United Kingdom.
Mutations in the chromodomain helicase DNA-binding protein 8 (CHD8) gene define a sub-type of monogenic autism, and in some cases, the mutations have been linked to schizophrenia and intellectual disabilities. In their latest research, the Zheng team used CRISPR/Cas to generate mutations in the CHD8 gene in human induced pluripotent stem cells (iPSCs). Human iPSCs are mature cells that have been reprogrammed into stem cells, which can then be transformed into other cell types. In this case, fibroblasts from skin biopsies donated by a healthy subject were used to generate the iPSCs.
The researchers then used the iPSCs with one mutated copy of CHD8 created by CRISPR/Cas to generate 3D brain organoids, following a previously established protocol. After 50 days in culture while adding various growth factors, inhibitors, and substrates at precise time points, the resulting aggregates of cells—brain organoids—are composed of neurons and structures resembling a first trimester telencephalon, and are about 1–2 millimetres in diameter.
The Zheng lab performed RNA-Seq analysis on four different sets of CHD8-mutated brain organoids and two different control sets, to determine which genes differ in their abundance levels between the two conditions. RNA-Seq allows for large-scale analyses and quantification of RNA products such as gene transcripts, and is usually used to uncover new targets in a disease model in a relatively unbiased manner. The team found that the differentially expressed genes between the CRISPR/Cas-generated CHD8 mutant and control brain organoids are similar to the differentially expressed genes between brain organoids generated from iPSCs derived from an autistic patient and from a healthy subject, validating that the CRISPR/Cas-generated CHD8 mutant brain organoids are a suitable model to study autism. The results also further confirmed some genetic overlap between autism and schizophrenia.
Interestingly, in both comparisons, the RNA-Seq analyses pointed to genes that are important for the development of inhibitory neurons. “This suggests a potential defect in inhibitory neuron functions in brains with autism spectrum disorder,” Dr. Zheng explained.
The potential of CRISPR/Cas in organoid systems
Although organoids can be cultured from iPSCs derived from biopsies of patients and healthy subjects, the genetic variations between any two people make it difficult to pinpoint the molecular mechanisms of a particular mutation. Now with the advances of CRISPR/Cas gene editing technology, a particular mutation can be generated in samples from one source to create control and mutated organoids, “in which the only difference between the control and the mutated sample is the mutation in the target gene—CHD8 in this case,” Dr. Zheng said of his lab’s new system to study autism.
The concept of combining CRISPR/Cas gene editing with organoid technology has also been used to explore other disease models, such as kidney disease using kidney organoids, and colorectal cancer using intestinal organoids. With the fast progression of both CRISPR/Cas gene editing and organoid technology, the intersection of these biomedical advances opens new avenues to study diseases and finding therapeutic targets.