CRISPR with high load capacity
Biotechnologists have learned how to embed giant fragments of DNA into genomes
Scientists at the Massachusetts Institute of Technology have developed a new PASTE gene editing tool based on CRISPR technology, which can cut out defective genes entirely and replace them in a more efficient and safe way. The method allows the introduction of giant DNA fragments several thousand nucleotides long into genomes. This is reported in an article published in the journal Nature Biotechnology (Yarnall et al., Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases).
PASTE combines CRISPR-Cas9 technology and integrase enzymes. CRISPR-Cas9 is a genetic "scissors" that were originally used by bacteria in the fight against viruses. The Cas9 protein cuts a double chain at a specific site of the target gene, which is determined by the guide RNA. If there is a separate piece of DNA nearby, then it is embedded in the place of the incision.
The disadvantage of CRISPR is that Cas9 produces double-stranded breaks that can lead to major DNA damage or even chromosomal rearrangements that cause cell death or its degeneration into cancer. Another limitation is the absence of activated DNA repair mechanisms in non-dividing cells, which are necessary for embedding the desired gene site. To solve this problem, the researchers used large serine integrases — enzymes of bacteriophages that embed viral DNA sequences into the genome of the host cell, namely into specific attB sites (from the English attachment) with a length of 30-50 nucleotides.
PASTE is based on the method of primed genome editing (Eng. prime editing). First, a complex consisting of a guide RNA and Cas9 binds to the target DNA sequence, after which a modified Cas9, called a nicase, makes a single-stranded incision. The reverse transcriptase enzyme uses an additional RNA chain associated with the complex as a template for synthesizing the desired DNA fragment at the incision site. Thus, the scientists managed to embed the attB site with a length of 46 base pairs, thus introducing a "landing pad" for serine integrase, which is active even in non-dividing cells.
The researchers demonstrated the integration of sequences up to 36,000 bases in three human cell lines, including lymphoblasts, primary T cells and non-dividing primary human hepatocytes. They tested a delivery system with 13 different genes, embedding them in nine different locations in the genome. The probability of successful insertion was 5-60 percent, while the method yielded very few unwanted inserts.
Currently, the authors are studying the possibility of using PASTE as a possible way to replace the defective gene that causes cystic fibrosis. This method can also be useful for the treatment of blood diseases caused by mutations, such as hemophilia, or Huntington's disease, a neurological disorder caused by an excess of nucleotide repeats.
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