PASTE: A Tool with Many Advantages and Great Potential
Ever since it was discovered that CRISPR could be used for genome editing, scientists across the world have been working on creating safe, effective ways to use this incredible tool to cure many different human diseases. Most recently, we have been presented with news of the development of another novel technique to expand the usefulness of CRISPR. MIT researchers wanted to create a system that can replace genes, rather than only correcting individual mutations – and they may have succeeded. The new tool is called PASTE (Programmable Addition via Site specific Targeting Elements), and it can essentially drag and drop an entire functional gene to replace the defective version, without inducing double-stranded DNA breaks. The team has integrated sequences as large as 36 kilobases in human cell lines, primary T cells, and non-dividing primary human hepatocytes in liver-humanized mice.
To overcome current limitations and expand the scope of genome editing, the team “married advances in programmable CRISPR-based gene editing, such as prime editing, with precise site-specific integrases”. To overcome current limitations and expand the scope of genome editing, the team “married advances in programmable CRISPR-based gene editing, such as prime editing, with precise site-specific integrases”.
Integrases, viral enzymes that allow a virus to insert its DNA into a bacterial genome, are a vital component of PASTE. The MIT team focused on phage serine integrases, as they can insert up to 50,000 base pairs. Serine integrases target specific DNA sequences called attachment (att) sites, which the integrase binds to, then integrates the genetic material.
While integrases are very difficult to reprogram to target other sites, coupling them with CRISPR-Cas9 enabled the team to more easily target the desired site in the genome for insertion of the landing site. As PASTE only knicks one strand of DNA, it avoids the undesirable outcomes of methods to insert long DNA sequences that create double-strand breaks. Furthermore, PASTE works in both non-dividing and dividing cells, whereas CRISPR-Cas9 only works in dividing cells as they have active DNA repair processes which are required to repair the cut made by CRISPR-Cas. Additionally, it can efficiently insert large sequences, rather than small insertions or deletions.
Instead of identifying and addressing each individual mutation of a disease, PASTE offers the potential to correct every disease-causing variant of a particular gene with one single blanket therapy for the disease, by inserting a full-length functional gene at native loci.
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