Nature Communications: Efficient and safe gene editing with Cas9TX in a mouse model of age-related macular degeneration

CRISPR-Cas9 is currently the most commonly used gene editing tool in the field, and has broad prospects
in basic scientific research and clinical applications.
However, while Cas9 completes the target site mutation, it will also cut the off-target site, and will cause chromosomal structural abnormalities such as chromosomal translocation and deletion of large chromosome fragments
.
In addition, in vivo gene editing therapy with adeno-associated virus (AAV) as the delivery vector, there is a phenomenon
of high-frequency insertion of AAV fragments.
The byproducts in these gene editing seriously threaten the stability of the genome, may lead to the cancerization of cells, and bring uncertainty
to the therapeutic outcome of gene editing.
By inhibiting the perfect repair product of Cas9 repeatedly cutting the target site, Cas9TX recently published by Hu Jiazhi's group can greatly reduce the frequency of chromosomal translocation during the modification of CAR-T, but whether Cas9TX can effectively reduce the production of these by-products in vivo gene editing scenarios that are more closely related to clinical application still needs further confirmation
.

The research group of Professor Hu Jiazhi of the School of Life Sciences of Peking University and the research group of Professor Yang Hui of the Institute of Neurology, Chinese Academy of Sciences in Shanghai jointly published the title "Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration" in Nature Communications Research papers
.
In the in vivo gene editing therapy model of age-related macular degeneration (AMD), this work quantitatively reveals for the first time the occurrence mode and frequency of chromosomal translocation and adeno-associated virus fragment insertion of CRISPR-Cas9 in the process of gene editing in vivo, and greatly reduces the production of these byproducts in the in vivo gene editing process by using the Cas9TX variant previously developed by the research group.
It provides important guidance for the clinical application of CRISPR-Cas9
.

Age macular degeneration is one of the leading causes of
blindness in older adults worldwide.
Among them, wet macular degeneration is mainly caused by abnormal angiogenesis behind the retina, and the injection of small molecules or antibodies that antagonize VEGFA protein that regulate angiogenesis is the mainstream treatment
of the disease.
However, repeated injections not only do not guarantee the efficiency of treatment, but also cause local complications
to the eyes.
Recently, CRISPR-Cas9-based gene editing technology has brought the dawn of treatment for the disease, by laser irradiation of mouse eyes to cause neovascularization to invade the retina to simulate macular degeneration, researchers further target Vegfa through Cas9, thereby eliminating the production of neovascularization once and for all, providing clinical operability for the treatment of the disease
.

Using PEM-seq, a high-throughput sequencing method developed by our group to comprehensively evaluate the safety of gene editing tools, this work first found that chromosomal translocations (frequency close to 1%) still form between targeted and off-target sites, between target sites and spontaneously generated DNA double-strand breaks in the genome (Figure A, below) in a mouse choroidoid hyperplasia editing model (targeting Vegfa site) using a dual AAV carrier packaging system as the delivery vehicle (Figure A
).
At the same time, the study also found that there is AAV fragment integration at the target site with a frequency of up to 40% (Figure B below
).
More importantly, these byproducts can be stable in the body for up to 12 weeks after gene editing, raising concerns about these byproducts
.

The results showed that Cas9TX could not only improve the editing efficiency of the target site, complete the treatment of the mouse choroidal hyperplasia model, but also greatly eliminate the chromosomal translocation generated at the target site (figure C below), it is worth mentioning that Cas9TX did not cause higher editing efficiency
at the off-target site.
More importantly, the work found that Cas9TX can also effectively reduce the integration of AAV fragments at the target site (Figure d).

It is reported that this is the first gene editing tool in the field that can reduce the insertion of AAV fragments in the gene editing process, which is of great significance
for clinical application.
Overall, this work not only shows that Cas9TX can be compatible with dual AAV delivery systems for gene editing in vivo, greatly reducing by-products in the gene editing process, but also shows the relative conservatism of DNA damage repair in vitro and in vivo, and uses this as a starting point to optimize the feasibility of gene editing safety
.

a.
PEM-seq to detect the frequency of chromosomal translocation after Cas9 editing of the Vegfa site in the mouse eye; b.
PEM-seq detects the frequency of AAV fragment insertion after Cas9 editing at the Vegfa site in the mouse eye; c.
Cas9TX significantly reduces the proportion of chromosomal translocations; d.
Cas9TX significantly reduces the proportion of AAV fragment insertions

Hu Jiazhi and Yang Hui are co-corresponding authors
of the paper.
Hong Jiaxu, Director of the Dry Eye Center of the Affiliated Eye, Ear, Nose and Throat Hospital of Fudan University in Shanghai, provided guidance
for this thesis.
Jianxing Yin, a 2022 doctoral student at Peking University's Frontier Interdisciplinary College, Karen Fang, a postdoctoral fellow at the Institute of Neurology, Shanghai Academy of Sciences, and Yanxia Gao, a postdoctoral fellow, are co-first authors
of the article.
Shaopeng Yuan, undergraduate students from the School of Life Sciences, Peking University, Ou Liqiong and Xin Changchang, doctoral students, and senior managers of Shanghai Huida Company, Weiwei Wu and Weiwei Wu, also made important contributions
to this work.