PRIORITIZATION OF EVOLUTIONARY MICROCEPHALY VARIANTS AND ISOGENIC LINE GENERATION AND CHARACTERIZATION HIGHLIGHTS <EM >KNL1</EM> AS A REGULATOR OF BRAIN SIZE

Human identity is mainly defined by cognitive skills grounded in the brain, which differentiates humans from other organisms, including evolutionarily close species. To unravel the evolution of brain along the human lineage implies identifying the few relevant variants from thousands of neutral mutations. We decided to focus on the 32 genes associated to primary microcephaly hereditary (MCPH), a neurodevelopmental disorder characterized by a reduced brain size in patients.
First, we performed a bioinformatical search for putative brain size-associated variants within the coding region of MCPH genes. We included variants fixed in humans and divergent from non-human primates (NHP), human common variants associated to brain size and reported pathogenic mutations. We found over 150 variants of interest. In KNL1, a gene encoding for a kinetochore protein, we prioritized 23 variants and were able to design CRISPR-cas9 guides to introduce the NHP variant for six of them. For the variant c.864G>T, p.K288N, we obtained one compound heterozygous knockout/knock-in (KO/KI) line with one truncating deletion in one allele and the edited NHP allele in the other. In addition, we successfully generated isogenic iPSC edited lines consisting of at least one heterozygous knockout (KO+/-) for three variants. We confirmed the absence of undesired off-target editions. We generated cortical brain organoids for these lines. The initial observations show an abnormal morphology of the KO/KI organoids compared to control and the KO+/- lines, that we are now replicating. These results point to the potential effect in brain size of human-specific variants within microcephaly-associated genes.