A team led by researchers from Broad Institute of MIT and Harvard, and the McGovern Institute for Brain Research at MIT, has characterized and engineered a new gene-editing system that can precisely and efficiently insert large DNA sequences into a genome.
The system, harnessed from cyanobacteria and called CRISPR-associated transposase (CAST), allows efficient introduction of DNA while reducing the potential error-prone steps in the process — adding key capabilities to gene-editing technology and addressing a long-sought goal for precision gene editing.
Precise insertion of DNA has the potential to treat a large swath of genetic diseases by integrating new DNA into the genome while disabling the disease-related sequence. To accomplish this in cells, researchers have typically used CRISPR enzymes to cut the genome at the site of the deleterious sequence, and then relied on the cell’s own repair machinery to stitch the old and new DNA elements together. However, this approach has many limitations.
Using Escherichia coli bacteria, the researchers have now demonstrated that CAST can be programmed to efficiently insert new DNA at a designated site, with minimal editing errors and without relying on the cell’s own repair machinery. The system holds potential for much more efficient gene insertion compared to previous technologies, according to the team.
The researchers are working to apply this editing platform in eukaryotic organisms, including plant and animal cells, for precision research and therapeutic applications.
The team molecularly characterized and harnessed CAST from two cyanobacteria, Scytonema hofmanni and Anabaena cylindrica, and additionally revealed a new way that some CRISPR systems perform in nature: not to protect bacteria from viruses, but to facilitate the spread of transposon DNA.
The work, appearing in Science, was led by first author Jonathan Strecker, a postdoctoral fellow at the Broad Institute; graduate student Alim Ladha at MIT; and senior author Feng Zhang, a core institute member at the Broad Institute, investigator at the McGovern Institute for Brain Research at MIT, the James and Patricia Poitras Professor of Neuroscience at MIT, and an associate professor at MIT, with joint appointments in the departments of Brain and Cognitive Sciences and Biological Engineering. Collaborators include Eugene Koonin at the National Institutes of Health.
A new role for a CRISPR-associated system
“One of the long-sought-after applications for molecular biology is the ability to introduce new DNA into the genome precisely, efficiently, and safely,” explains Zhang. “We have worked on many bacterial proteins in the past to harness them for editing in human cells, and we’re excited to further develop CAST and open up these new capabilities for manipulating the genome.”
To expand the gene-editing toolbox, the team turned to transposons. Transposons (sometimes called “jumping genes”) are DNA sequences with associated proteins — transposases — that allow the DNA to be cut-and-pasted into other places.
Most transposons appear to jump randomly throughout the cellular genome and out to viruses or plasmids that may also be inhabiting a cell. However, some transposon subtypes in cyanobacteria have been computationally associated with CRISPR systems, suggesting that these transposons may naturally be guided towards more-specific genetic targets. This theorized function would be a new role for CRISPR systems; most known CRISPR elements are instead part of a bacterial immune system, in which Cas enzymes and their guide RNA will target and destroy viruses or plasmids.
In this paper, the research team identified the mechanisms at work and determined that some CRISPR-associated transposases have hijacked an enzyme called Cas12k and its guide to insert DNA at specific targets, rather than just cutting the target for defensive purposes.
“We dove deeply into this system in cyanobacteria, began taking CAST apart to understand all of its components, and discovered this novel biological function,” says Strecker, a postdoctoral fellow in Zhang’s lab at the Broad Institute. “CRISPR-based tools are often DNA-cutting tools, and they’re very efficient at disrupting genes. In contrast, CAST is naturally set up to integrate genes. To our knowledge, it’s the first system of this kind that has been characterized and manipulated.”
Harnessing CAST for genome editing
Once all the elements and molecular requirements of the CAST system were laid bare, the team focused on programming CAST to insert DNA at desired sites in E. coli.
“We reconstituted the system in E. coli and co-opted this mechanism in a way that was useful,” says Strecker. “We reprogrammed the system to introduce new DNA, up to 10 kilobase pairs long, into specific locations in the genome.”
The team envisions basic research, agricultural, or therapeutic applications based on this platform, such as introducing new genes to replace DNA that has mutated in a harmful way — for example, in sickle cell disease. Systems developed with CAST could potentially be used to integrate a healthy version of a gene into a cell’s genome, disabling or overriding the DNA causing problems.
Alternatively, rather than inserting DNA with the purpose of fixing a deleterious version of a gene, CAST may be used to augment healthy cells with elements that are therapeutically beneficial, according to the team. For example, in immunotherapy, a researcher may want to introduce a “chimeric antigen receptor” (CAR) into a specific spot in the genome of a T cell — enabling the T cell to recognize and destroy cancer cells.
“For any situation where people want to insert DNA, CAST could be a much more attractive approach,” says Zhang. “This just underscores how diverse nature can be and how many unexpected features we have yet to find.”
The Latest on: CRISPR-associated transposase
via Google News
The Latest on: CRISPR-associated transposase
- Inserting DNA with CRISPRon July 4, 2019 at 1:01 pm
Medical Center Drive, Ann Arbor, MI 48109, USA. CRISPR-associated transposase (CAST)–mediated DNA insertion is guided by CRISPR-Cas12k. The inserted transposon can include cargo DNA for genetic ... […]
- How transposons improve CRISPR efficiencyon June 25, 2019 at 1:45 pm
The clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) endonuclease system ... which encode for an enzyme called transposase that acts in a “cut and paste” ... […]
- New gene-editing system uses ‘jumping genes’ to insert DNA without cutting themon June 15, 2019 at 3:06 am
They then inserted the new DNA sequences without the need to cut anything. The team called the new system as CRISPR-associated transposase (CAST). “We dove deeply into this system in cyanobacteria, ... […]
- Scientists discover new error-reducing gene-editing systemon June 13, 2019 at 6:29 am
The system that the team developed uses cyanobacteria and is called CRISPR-associated transposase (CAST). The new method allows for the efficient introduction of DNA and reduced potential for error in ... […]
- New CRISPR-based system inserts new DNA without cutting and pastingon June 12, 2019 at 8:31 pm
The new system was named CRISPR-associated transposase (CAST). "We dove deeply into this system in cyanobacteria, began taking CAST apart to understand all of its components, and discovered this novel ... […]
- CRISPR Jumps in New Directionon June 12, 2019 at 1:34 pm
He adds that “this is an interesting point of distinction with the CRISPR-associated transposase described by Zhang and colleagues, which exhibits a relatively low degree of accuracy and promiscuosly ... […]
- CRISPR-associated transposons able to insert custom genes into DNA without cutting iton June 7, 2019 at 6:30 am
In this new effort, the researchers have found a way to use CRISPR in conjunction with another protein to edit a strand of DNA without cutting it—they are calling it CRISPR-associated transposase ... […]
- New Approach to CRISPR Could Yield Even Better Gene Editingon June 6, 2019 at 2:30 pm
Instead of chopping through both strands of DNA to do its work, the new method, called a CRISPR-associated transposase (CAST), simply inserts a gene, no double-strand slicing required. The advance ... […]
- ‘Jumping genes’ could help CRISPR replace disease-causing DNA, study findson June 6, 2019 at 11:01 am
Zhang calls the system “CRISPR-associated transposase,” or CAST. He and one of his co-authors, Jonathan Strecker, have filed for a patent on it. CAST seems to have no such problem inserting DNA. When ... […]
- Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattleon December 18, 2017 at 4:00 pm
To repair the mutated IARS gene, we designed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9 ... cassette with a piggyBac transposase recognition ... […]
via Bing News