Nature sub-issue: Peking University Hu Jiazhi's team developed a safer Cas9 variant Cas9TX

CRISPR-Cas nucleases are currently the most widely used gene editing tools, and have broad application prospects in both basic scientific research and clinical applications
.

However, in addition to off-target activity, CRISPR-Cas also produces chromosomal structural abnormalities such as chromosomal translocations and deletions of large chromosomal segments.

CRISPR-Cas nucleases are currently the most widely used gene editing tools, and have broad application prospects in both basic scientific research and clinical applications

In October 2021 , a lymphoma patient had a reduction in all blood cell lines following treatment with LymphomaAllogene's gene-edited next-generation generic CAR-T CAR-T candidate ALLO-501A , biopsy analysis It was found that CAR-T cells with chromosomal translocation appeared in his body .
The FDA has suspended all Allogene CAR -T clinical trials due to the serious potential risk of chromosomal translocations that can lead to cancer .

With the continuous popularization of the clinical application of gene editing, the number of patients with uncontrollable chromosomal translocations in the edited cells will inevitably increase gradually
.

However, due to the lack of understanding of the mechanism of abnormal chromosome structure in the process of gene editing, there is no effective strategy to inhibit the occurrence of such by-products in the field

With the continuous popularization of the clinical application of gene editing, the number of patients with uncontrollable chromosomal translocations in the edited cells will inevitably increase gradually

On March 8 , 2022 , Hu Jiazhi's research group from Peking University School of Life Sciences and Peking University-Tsinghua Life Science Joint Center published a research paper titled: Cas9 exo-endonuclease eliminates chromosomal translocations during genome editingin thejournal Nature Communications[ 1 ] .

This study revealed the molecular mechanism of chromosomal translocation during gene editing, and targeted the design and development of a novel gene editing enzyme, Cas9TX , with greatly improved safety

It is worth mentioning that the research team applied Cas9TX to the production of next-generation universal CAR-T cells and found that Cas9TX reduced the chromosomal translocation between targeted sites to the background without affecting the killing efficiency of CAR-T .
level .

Based on PEM-seq [ 2 ], a method for comprehensively evaluating the safety of gene editing developed by Hu Jiazhi's group in 2019 , the research team first evaluated the safety of the new generation of CAR-T technology .

A detailed operating guide for the PEM-seq method will also be published in the Cell series of STAR protocols in 2022 [ 3 ] .

Based on PEM-seq [ 2 ], a method for comprehensively evaluating the safety of gene editing developed by Hu Jiazhi's group in 2019 , the research team first evaluated the safety of the new generation of CAR-T technology .
A detailed operating guide for the PEM-seq method will also be published in the STAR protocols of the Cell series in 2022 [ 3 ] .

The construction of next-generation universal CAR-T cells combines gene editing technology to knock out TRAC , B2M , PDCD1 and other genes while producing CAR-T cells, which can reduce the immune rejection between donor and recipient , Realize the possibility of allogeneic transplantation, and at the same time improve the killing ability of CAR-T cells .

In this study, the authors first used PEM-seq to detect the probability of chromosomal structural abnormalities in the new generation of CAR-T cells.
The analysis showed that chromosomal translocations commonly occurred between multiple Cas9 target sites, and the proportion accounted for About 1% of editor-in-chief events .

The construction of next-generation universal CAR-T cells combines gene editing technology to knock out TRAC , B2M , PDCD1 and other genes while producing CAR-T cells, which can reduce the immune rejection between donor and recipient , Realize the possibility of allogeneic transplantation, and at the same time improve the killing ability of immune CAR-T cells .
In this study, the authors first used PEM-seq to detect the probability of chromosomal structural abnormalities in the new generation of CAR-T cells.
The analysis showed that chromosomal translocations commonly occurred between multiple Cas9 target sites, and the proportion accounted for About 1% of editor-in-chief events .
This means that in CAR-T cell therapy, for every 10^8 CAR-T cells reinfused into a patient, more than 10^6 cells carry chromosomal translocations .

It is worth noting that previous studies on lymphoma have shown that there are transcriptional enhancers near TRAC that can greatly promote gene expression, which can increase the expression level of nearby proto-oncogenes after translocation .

The study further pointed out that Cas9 high-fidelity variants commonly used in the field to eliminate off-target activity cannot effectively inhibit the occurrence of chromosomal translocations between Cas9 -targeted sites (Figure 1 ) .

It is worth noting that previous studies on lymphoma have shown that there are transcriptional enhancers near TRAC that can greatly promote gene expression, which can increase the expression level of nearby proto-oncogenes after translocation .
The study further pointed out that Cas9 high-fidelity variants commonly used in the field to eliminate off-target activity cannot effectively inhibit the occurrence of chromosomal translocations between Cas9 -targeted sites (Figure 1 ) .

Figure 1.

  Chromosomal translocations are common in the production of next-generation CAR-T cells

Figure 1.
  Chromosomal translocations are common in the production of next-generation CAR-T cells

In order to explore the regularity of chromosomal translocations during gene editing, this study carried out a detailed analysis of PEM-seq data.

The authors unexpectedly found that there is a clear translocation connection between the Cas9 target site and the off-target site.

In order to explore the regularity of chromosomal translocations during gene editing, this study carried out a detailed analysis of PEM-seq data.
The authors unexpectedly found that there is a clear translocation connection between the Cas9 target site and the off-target site.

Figure 2.
Repeated cleavage of perfect repair products by Cas9 results in massive chromosomal translocations

Figure 2.
Repeated cleavage of perfect repair products by Cas9 results in massive chromosomal translocations

The authors then attempted to couple an exonuclease to Cas9 to reduce the proportion of perfect repair products by modifying the DNA break ends during the editing process , thereby inhibiting repeated cleavage by Cas9 (Fig.
2 bottom) .
By screening a variety of exonuclease, this study finally coupled the optimized human TREX2 protein with Cas9 to generate the exonuclease Cas9TX .
PEM-seq analysis showed that Cas9TX can indeed inhibit the repeated cleavage of Cas9 , and almost eliminated the Generation of chromosomal translocations .
Then the authors verified through whole-genome sequencing and other means that Cas9TX does not cause non-specific cleavage of the genome, it can stably and effectively inhibit the generation of chromosomal translocations, and achieve the same base editor that causes DNA single-strand breaks.
the same level .
In addition, the researchers found that Cas9TX can also greatly reduce the generation of large chromosomal deletions .

The authors then attempted to couple an exonuclease to Cas9 to reduce the proportion of perfect repair products by modifying the DNA break ends during the editing process , thereby inhibiting repeated cleavage by Cas9 (Fig.
2 bottom) .
By screening a variety of exonuclease, this study finally coupled the optimized human TREX2 protein with Cas9 to generate the exonuclease Cas9TX .
PEM-seq analysis showed that Cas9TX can indeed inhibit the repeated cleavage of Cas9 , and almost eliminated the Generation of chromosomal translocations .
Then the authors verified through whole-genome sequencing and other means that Cas9TX does not cause non-specific cleavage of the genome, it can stably and effectively inhibit the generation of chromosomal translocations, and achieve the same base editor that causes DNA single-strand breaks.
the same level .
In addition, the researchers found that Cas9TX can also greatly reduce the generation of large chromosomal deletions .

At the same time, while inhibiting the occurrence of abnormal chromatin structure, Cas9TX can maintain the same level or slightly higher level of gene editing as Cas9 .
It is also worth pointing out that the mutated TREX2 is only 236 amino acids and does not significantly increase the size of Cas9 (Figure 3 ) .

At the same time, while inhibiting the occurrence of abnormal chromatin structure, Cas9TX can maintain the same level or slightly higher level of gene editing as Cas9 .
It is also worth pointing out that the mutated TREX2 is only 236 amino acids and does not significantly increase the size of Cas9 (Figure 3 ) .

Figure 3.
Cas9TX virtually eliminates the generation of chromosomal translocations during gene editing

Figure 3.
Cas9TX virtually eliminates the generation of chromosomal translocations during gene editing

The research team further applied Cas9TX to the preparation of next-generation universal CAR-T cells and found that Cas9TX reduced the chromosomal translocation between targeted sites to the background level without affecting the killing effect of CAR-T (Figure 4 ).
) .

The research team further applied Cas9TX to the preparation of next-generation universal CAR-T cells and found that Cas9TX reduced the chromosomal translocation between targeted sites to the background level without affecting the killing effect of CAR-T (Figure 4 ).
) .

Figure 4.
Cas9TX improves the safety of CAR-T cell production

Figure 4.
Cas9TX improves the safety of CAR-T cell production

In addition to CAR-T cells making this ex vivo gene editing case, the research team also applied Cas9TX to a mouse model of age-related macular degeneration ( AMD ), an in vivo eye disease model , and they found that Cas9TX was effective in eliminating chromosomal translocations At the same time, it can also greatly inhibit the integration of AAV vectors .
All in all, the Cas9TX shows amazing results .

In addition to CAR-T cells making this ex vivo gene editing case, the research team also applied Cas9TX to a mouse model of age-related macular degeneration ( AMD ), an in vivo eye disease model , and they found that Cas9TX was effective in eliminating chromosomal translocations At the same time, it can also greatly inhibit the integration of AAV vectors .
All in all, the Cas9TX shows amazing results .

Researcher Hu Jiazhi of Peking University is the corresponding author of this article
.
The study was strongly supported by Liu Tao, Peking University School of Medicine, Peking University researchers Li Xiangying and Wei Ping, and Dr.
Xiao Bingbing, Department of Obstetrics and Gynecology, Peking University First Hospital Obstetrics and Children's
Hospital .
Yin Jianxing, Lu Rusen and Xin Changchang, doctoral students in the laboratory of Researcher Hu Jiazhi, are the co-first authors of this article, which also received great help from other co-authors
.

Researcher Hu Jiazhi of Peking University is the corresponding author of this article
.
The study was strongly supported by Liu Tao, Peking University School of Medicine, Peking University researchers Li Xiangying and Wei Ping, and Dr.
Xiao Bingbing, Department of Obstetrics and Gynecology, Peking University First Hospital Obstetrics and Children's
Hospital .
Yin Jianxing, Lu Rusen and Xin Changchang, doctoral students in the laboratory of Researcher Hu Jiazhi, are the co-first authors of this article, which also received great help from other co-authors
.
child

Paper link:

Paper link:

1.
https:// class="s2">1.
https:// class="p3">2.
https:// class="s2">2.
https:// class="p3">3.
https://doi.
org/10.
1016/j.
xpro.
2021.
101088

3.
https://doi.
org/10.
1016/j.
xpro.
2021.
101088 Leave a message here