crisprcas9-mediated-targeted-integration-vivo-using-homology-mediated

CRISPR/Cas9-mediated Targeted Integration <em>In Vivo</em> Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Shanghai Tech university

Shanghai Tech University

Hepatocytes are functionally heterogeneous and are divided into two distinct populations based on their metabolic zonation: the periportal and pericentral hepatocytes. During liver injury and regeneration, the cellular dynamics of these two distinct populations remain largely elusive. Here we show that major facilitator super family domain containing 2a (Mfsd2a), previously known to maintain blood-brain barrier function, is a periportal zonation marker. By genetic lineage tracing of Mfsd2a(+) periportal hepatocytes, we show that Mfsd2a(+) population decreases during liver homeostasis. Nevertheless, liver regeneration induced by partial hepatectomy significantly stimulates expansion of the Mfsd2a(+) periportal hepatocytes. Similarly, during chronic liver injury, the Mfsd2a(+) hepatocyte population expands and completely replaces the pericentral hepatocyte population throughout the whole liver. After injury recovery, the adult liver re-establishes the metabolic zonation by reprogramming the Mfsd2a(+)-derived hepatocytes into pericentral hepatocytes. The evidence of entire zonation replacement during injury increases our understanding of liver biology and disease.

Mfsd2a+ hepatocytes repopulate the liver during injury and regeneration.

Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR)-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ)-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold) than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fahliver failure mice, offering an efficient approach for precisely targeted gene therapies.

CRISPR/Cas9 - Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms.

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.

Pengyu Huang

School of Life Science and Technology

Shanghai Tech university

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy