Crop Biotech Update

New CRISPR Tool Allows Precise Inserts of Large DNA Sequences in Targeted Sites

December 7, 2022

Massachusetts Institute of Technology scientists are one step closer to achieving the dream of programmable insertion of DNA thanks to a new CRISPR-based tool that they developed.

The new tool is called PASTE, which stands for Programmable Addition via Site-specific Targeting Elements. The concept came from the desire to make a tool that could cut out a defective gene and replace it with a new one, without inducing any double-stranded DNA breaks. To do this, the scientists turned to serine integrases which are known to be able to insert huge chunks of DNA. These enzymes focus on attachment sites in the genome that serve as landing pads and bind to it to integrate their DNA payload.

PASTE combines the precise targeting of CRISPR-Cas9 and the integrases. The tool includes a Cas9 enzyme that cuts at a specific genome site using a strand of RNA guide that binds to that site. Any site in the genome can be targeted for insertion of the landing site. PASTE also allows one DNA strand to be added first using fused reverse transcriptase followed by its complementary strand. This reduces the chances of causing double-stranded breaks during the process.

PASTE was found to have a success rate ranging from five to 60 percent and yielded very few unwanted indels. The tool helps scientists reduce the risk of chromosomal rearrangements or large-scale chromosome arm deletions. With further investigations, the tool can be useful in treating diseases caused by defective genes with a large number of mutations like cystic fibrosis.

Learn more from MIT News.

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