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 ...

Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.

Homology-mediated end joining-based targeted integration using CRISPR/Cas9.

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.

Liver regeneration (LR) happens after various types of injuries. Unlike the well-studied LR caused by partial hepatectomy (PHx), there is accumulating evidence suggesting that LR during other injuries may result from unknown mechanisms. In this study, we found that insulin-like growth factor 2 (IGF-2) was drastically induced following the liver injuries caused by tyrosinemia or long-term treatments of CCl. However, this was not observed during the early phase of acute liver injuries after PHx or single treatment of CCl. Remarkably, most IGF-2-expressing hepatocytes were located at the histological area around the central vein of the liver lobule after the liver injuries caused either in fumarylacetoacetate hydrolase-deficient mice or in CClchronically treated mice. Hepatocyte proliferation in vivo was significantly promoted by induced IGF-2 overexpression, which could be inhibited by adeno-associated virus-delivered IGF-2 short hairpin RNAs or linsitinib, an inhibitor of IGF-2 signaling. Proliferating hepatocytes in vivo responded to IGF-2 through both insulin receptor and IGF-1 receptor. IGF-2 also significantly promoted DNA synthesis of primary hepatocytes in vitro. More interestingly, the significantly induced IGF-2 was also found to colocalize with glutamine synthetase in the region enriched with proliferating hepatocytes for the liver samples from patients with liver fibrosis.

Pericentral hepatocytes produce insulin-like growth factor-2 to promote liver regeneration during selected injuries in mice.

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.

Article	Year
Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell research\| PubMed ID: 28524166	2017
CRISPR/Cas9 - Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms. EBioMedicine\| PubMed ID: 28527830	2017
Pericentral hepatocytes produce insulin-like growth factor-2 to promote liver regeneration during selected injuries in mice. Hepatology (Baltimore, Md.)\| PubMed ID: 28653763	2017
In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice. Nature neuroscience\| PubMed ID: 29335603	2018

Junlai Liu

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