Breakthrough in Gene Editing CRISPR–Cas systems: First example of a fully programmable, RNA-guided integrase and lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.
Reporter: Aviva Lev-Ari, PhD, RN
CRISPR alternatives for editing genes without cutting: CRISPR 12, 12a, 13, 14 – Alternative Techniques to CRISPR/Cas9
- Alternative to CRISPR/Cas9 – CAST (CRISPR-associated transposase) – A New Gene-editing Approach for Insertion of Large DNA Sequences into a Genome developed @BroadInstitute @MIT @Harvard
Reporter: Aviva Lev-Ari, PhD, RN
- Vertex Pharmaceuticals agreed to pay $420 million to acquire Exonics and to expand its partnership with CRISPR Therapeutics. The deal sets in motion a planto use CRISPR to treat Duchenne muscular dystrophy and myotonic dystrophy type 1.
- In May, a team at the Fred Hutchinson Cancer Research Center described a method developed there to use gold nanoparticles to carry CRISPR components into cells and to use the Cas12a enzyme to make cleaner cuts than Cas9 typically does.
- A UC Berkeley spinoff, GenEdit, is also developing a gold-based CRISPR system.
- Other recently proposed ideas for improving CRISPR include attaching a hairpin-like guide to RNA to improve the accuracy of DNA cuts and adding an on-off switch to Cas9 enzymes to ensure they can’t make edits anywhere other than the targeted sites.
- The next step for Sternberg’s team at Columbia is to test the INTEGRATE technology in mammalian cells. They believe the technique could eventually be applied to a variety of products, such as gene therapies and engineered crops.
Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration
Nature (2019)
Abstract
Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a remarkable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR–Cas systems to catalyze RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in E. coli requires CRISPR- and transposon-associated molecular machineries, including a novel co-complex between Cascade and the transposition protein TniQ. Donor DNA integration occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep sequencing experiments reveal highly specific, genome-wide DNA integration across dozens of unique target sites. This work provides the first example of a fully programmable, RNA-guided integrase and lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.
A CRISPR alternative for editing genes without cutting
The researchers are using a “jumping gene,” or transposon, from a bacterium called Vibrio cholerae. The transposon is able to insert itself into different regions of the genome and can be programmed to carry any DNA sequence to any site. Therefore their technology, which they dubbed INTEGRATE, acts less like molecular scissors and more like molecular glue, they explained in the journal Nature.
“Rather than introduce DNA breaks and rely on the cell to repair the break, INTEGRATE directly inserts a user-defined DNA sequence at a precise location in the genome, a capability that molecular biologists have sought for decades,” said senior author Sam Sternberg, Ph.D., assistant professor of biochemistry and molecular biophysics at Columbia, in a statement. Sternberg recently joined Columbia after a stint working in the lab of CRISPR pioneer Jennifer Doudna at the University of California, Berkeley.
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