Precisely controlling endogenous protein dosage in hPSCs and derivatives to model FOXG1 syndrome

Wenliang Zhu,Fan Mo,Yihui Wu,Zhaoqian Teng,Wei Li,Mengqi Li,Qi Zhou,Boya Zhang,Baoyang Hu,Tingwei Mi

doi:10.1038/s41467-019-08841-7

Abstract

Dosage of key regulators impinge on developmental disorders such as FOXG1 syndrome. Since neither knock-out nor knock-down strategy assures flexible and precise protein abundance control, to study hypomorphic or haploinsufficiency expression remains challenging. We develop a system in human pluripotent stem cells (hPSCs) using CRISPR/Cas9 and SMASh technology, with which we can target endogenous proteins for precise dosage control in hPSCs and at multiple stages of neural differentiation. We also reveal FOXG1 dose-dependently affect the cellular constitution of human brain, with 60% mildly affect GABAergic interneuron development while 30% thresholds the production of MGE derived neurons. Abnormal interneuron differentiation accounts for various neurological defects such as epilepsy or seizures, which stimulates future innovative cures of FOXG1 syndrome. By means of its robustness and easiness, dosage-control of proteins in hPSCs and their derivatives will update the understanding and treatment of additional diseases caused by abnormal protein dosage.

Highlights

Dosage of key regulators impinge on developmental disorders such as FOXG1 syndrome
Small molecule-assisted shut-off (SMASh) is a technique in which proteins are fused to a self-removing degron that allows reversible and dose-dependent shut-off by administration of small molecules[35]
In order to study dosage related diseases such as FOXG1 syndrome, we adapt small molecule-assisted shut-off (SMASh) system for human pluripotent stem cells (hPSCs) via CRISPR mediated genome editing to precisely control the dosage of endogenous proteins

Summary

Introduction

Dosage of key regulators impinge on developmental disorders such as FOXG1 syndrome. Since neither knock-out nor knock-down strategy assures flexible and precise protein abundance control, to study hypomorphic or haploinsufficiency expression remains challenging. FOXG1 syndrome exhibits variable symptoms such as autism spectrum disorder (ASD), epilepsy, microcephaly (congenital or postnatal), severe intellectual disability, abnormal or involuntary movements, and unexplained episodes of crying[16,17,18,19,20]. Such diverse spectrum of neurological manifestations indicate that in patients of FOXG1 syndrome excitatory and inhibitory cortical neurons are variably constituted. We engineer hPSCs with SMASh tagged protein using CRISPR/Cas[9] for precise dosage control, with which we can model protein dosage related disease such as FOXG1 syndrome

Methods

Results

Conclusion