Human gene editing and in situ sequencing of neuronal microcircuit arrays

  • Awarded: 2015
  • Award Type: Targeted: Functional Screen of Autism-Associated Variants
  • Award #: 368485

The brain is an exquisitely complex network, and the precise development of neuronal connections into the appropriate circuitry is crucial in determining brain function. Malformation of these connections during prenatal and early postnatal development can lead to neurological deficits, including intellectual disability, autism and schizophrenia. While inappropriate circuit formation has been suggested to be a critical deficit in autism, precisely whether and how various autism-related gene mutations lead to such defects remains unclear.

Over the past several years, a wealth of information about the potential cellular and genetic causes of autism has come about, as a result of next-generation sequencing of children with autism and their unaffected family members, as part of the Simons Simplex Collection (SSC). This wealth has also proven a challenge, as researchers attempt to move beyond identification of genetic variants toward an understanding of the biological and physiological consequences of these mutations for the disease. George Church and his team at Harvard Medical School plan to assess how these gene mutations affect neuronal circuitry, using a simplified neuronal circuit system combined with state-of-the art gene knock-out technology.

Church and his team will create a microcircuit culture system based on induced pluripotent stem-cell-derived neurons, culturing small (one-to-three cell) circuits on microscope slides. The team will then use the CRISPR/CAS9 system to virally deliver a library of short guide RNAs (sgRNAs) targeted to genes identified from the SSC. This approach will effectively ‘knock out’ genes linked to autism, allowing the researchers to track and monitor how loss of these genes alters cellular and circuit phenotypes in their culture system. Using a variety of cellular and optical tools, the team will then screen for defects in a wide variety of neuronal functions, including neuronal development, maturation, connectivity and activity. Genes showing clear cellular or circuit phenotypes will be further validated using traditional electrophysiological analyses compatible with the microcircuit culture system.

A major advantage that this platform affords is the ability to screen mutations in human cells, hundreds to thousands at a time, in an environment with a highly controlled and replicable cellular context. The ability to do so takes advantage of several recent technical advances, many developed by Church and his team. Successful implementation of this screening microcircuit technology will allow researchers to fairly rapidly functionally characterize a large number of autism-related genes for cellular and circuit defects.

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