Using laboratory-evolved versions of CRISPR-associated transposes (CASTs) from bacteria, scientists at the Broad Institute and Columbia University were able to insert healthy genes into human cells efficiently enough for potential therapeutic applications. Full details about the system, dubbed evoCAST, are provided in a new Science paper titled, “Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase.”
This research comes out of the laboratories of David Liu, PhD, and Samuel Sternberg, PhD. Liu is a professor and the director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad as well as a professor at Harvard University. Sternberg is an associate professor in the department of biochemistry and molecular biophysics at Columbia University. Both Liu and Sternberg are also Howard Hughes Medical Institute Investigators.
CASTs are naturally occurring enzymes that utilize CRISPR machinery to move and integrate large stretches of DNA in bacterial genomes without creating the free double-strand breaks that can often lead to unwanted editing byproducts. The engineered CASTs developed by Liu and Sternberg’s groups can insert DNA sequences thousands of bases long into target sites of therapeutic interest. And they are hundreds of times more efficient than the natural CAST systems that have been reported to date, according to the scientists.
CASTs have so far shown minimal activity in mammalian cells. “In this work, we discovered that a key bottleneck for CAST in human cells was its limited transposition activity,” said Isaac Witte, a graduate student in Liu’s lab and a co-first author on the work. By addressing this bottleneck through laboratory evolution, “CAST can excel as a genome editing tool in human cells.”
The results described in the Science paper build on earlier work done by Sternberg and other scientists, including Feng Zhang, PhD, a professor of neuroscience, brain and cognitive sciences, and biological engineering at the Massachusetts Institute of Technology. In 2019, they characterized CASTs in bacteria. However, when Sternberg’s lab used these enzymes to edit human cells, they found that editing occurred in about 0.1% of cells, much too low to be used as a potential therapeutic.
To address this problem, Sternberg and Liu used the phage-assisted continuous evolution (PACE) system developed in the Liu lab. Using PACE, the scientists evolved a CAST system from Pseudoalteromonas bacteria. After hundreds of rounds of evolution, they generated an “evolved variant of the CAST transposase protein TnsB,” according to the paper. They then combined “this evolved TnsB with other PACE-evolved and rationally engineered CAST components to yield evoCAST, a system optimized for human-cell integration activity.”
The developers claim that EvoCast is over four hundred times more efficient than wild-type CAST. They demonstrated that it supports large DNA cargoes that are more than 10 kb long. In experiments with human cells, the scientists used evoCAST to install genes relevant for diseases such as Fanconi anemia and phenylketonuria. They have also used it to improve CAR-T immunotherapies with efficiency between 10 and 20 percent.
Liu compared evoCAST to eePASSIGE, another gene-editing technique developed in his lab that is designed to insert or substitute genes or gene-sized DNA segments in human cells. He noted that while eePASSIGE is more efficient, evoCAST edits with higher purity. It also installs genes in a single step, which could help scientists apply it more quickly than eePASSIGE, which uses two steps to integrate new DNA and requires a prime editor and a recombinase enzyme.
EvoCAST is not quite ready for use in human therapies. For their next steps, the scientists are making improvements to evoCAST to get it to the point where it can be used therapeutically. They also plan to apply the CAST PACE platform to other types of CAST systems in nature.
“Mobile genetic elements like CAST offer a glimpse into nature’s ingenuity in programmable genome manipulation,” said Sternberg. “With evoCAST, we’ve shown how laboratory evolution can transform these naturally occurring systems into powerful tools for therapeutic gene insertion—and we believe we’ve only scratched the surface of what will be possible in the future.”
The post EvoCAST Harnesses CRISPR-Linked Bacterial Transposes to Insert Genes Into Human Cells appeared first on GEN - Genetic Engineering and Biotechnology News.
This research comes out of the laboratories of David Liu, PhD, and Samuel Sternberg, PhD. Liu is a professor and the director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad as well as a professor at Harvard University. Sternberg is an associate professor in the department of biochemistry and molecular biophysics at Columbia University. Both Liu and Sternberg are also Howard Hughes Medical Institute Investigators.
CASTs are naturally occurring enzymes that utilize CRISPR machinery to move and integrate large stretches of DNA in bacterial genomes without creating the free double-strand breaks that can often lead to unwanted editing byproducts. The engineered CASTs developed by Liu and Sternberg’s groups can insert DNA sequences thousands of bases long into target sites of therapeutic interest. And they are hundreds of times more efficient than the natural CAST systems that have been reported to date, according to the scientists.
CASTs have so far shown minimal activity in mammalian cells. “In this work, we discovered that a key bottleneck for CAST in human cells was its limited transposition activity,” said Isaac Witte, a graduate student in Liu’s lab and a co-first author on the work. By addressing this bottleneck through laboratory evolution, “CAST can excel as a genome editing tool in human cells.”
The results described in the Science paper build on earlier work done by Sternberg and other scientists, including Feng Zhang, PhD, a professor of neuroscience, brain and cognitive sciences, and biological engineering at the Massachusetts Institute of Technology. In 2019, they characterized CASTs in bacteria. However, when Sternberg’s lab used these enzymes to edit human cells, they found that editing occurred in about 0.1% of cells, much too low to be used as a potential therapeutic.
To address this problem, Sternberg and Liu used the phage-assisted continuous evolution (PACE) system developed in the Liu lab. Using PACE, the scientists evolved a CAST system from Pseudoalteromonas bacteria. After hundreds of rounds of evolution, they generated an “evolved variant of the CAST transposase protein TnsB,” according to the paper. They then combined “this evolved TnsB with other PACE-evolved and rationally engineered CAST components to yield evoCAST, a system optimized for human-cell integration activity.”
The developers claim that EvoCast is over four hundred times more efficient than wild-type CAST. They demonstrated that it supports large DNA cargoes that are more than 10 kb long. In experiments with human cells, the scientists used evoCAST to install genes relevant for diseases such as Fanconi anemia and phenylketonuria. They have also used it to improve CAR-T immunotherapies with efficiency between 10 and 20 percent.
Liu compared evoCAST to eePASSIGE, another gene-editing technique developed in his lab that is designed to insert or substitute genes or gene-sized DNA segments in human cells. He noted that while eePASSIGE is more efficient, evoCAST edits with higher purity. It also installs genes in a single step, which could help scientists apply it more quickly than eePASSIGE, which uses two steps to integrate new DNA and requires a prime editor and a recombinase enzyme.
EvoCAST is not quite ready for use in human therapies. For their next steps, the scientists are making improvements to evoCAST to get it to the point where it can be used therapeutically. They also plan to apply the CAST PACE platform to other types of CAST systems in nature.
“Mobile genetic elements like CAST offer a glimpse into nature’s ingenuity in programmable genome manipulation,” said Sternberg. “With evoCAST, we’ve shown how laboratory evolution can transform these naturally occurring systems into powerful tools for therapeutic gene insertion—and we believe we’ve only scratched the surface of what will be possible in the future.”
The post EvoCAST Harnesses CRISPR-Linked Bacterial Transposes to Insert Genes Into Human Cells appeared first on GEN - Genetic Engineering and Biotechnology News.