Prime Editing – An Exciting Combination of Natural and Engineered RNA-guided Genome Editing

How CRISPR Prime or Prime Editing Works

Prime Editing is a novel variation on CRISPR systems which expands the guide RNA’s responsibility to serve two purposes:

1. to guide Cas9 to a targeted genomic location, and
2. to serve as an RNA template to copy new sequences into the DNA genome (Anzalone, 2019).

In addition to maintaining the elegantly simple two-molecule CRISPR approach (Cas9 + gRNA), Prime Editing employs an editing mechanism that operates independent of the host cell’s homology-directed repair (HDR) machinery which is typically employed to copy information from donor DNA templates. Many human cell types are not competent to perform HDR at high efficiency; thus, Prime Editing stands to by-pass HDR and enable high-frequency edits in a broad range of cell types. Lastly, Prime Editing provides a solution for a lesser-known, but very serious problem for many CRISPR-HDR projects: toxic cellular responses to double-stranded DNA donor templates. AAV approaches have enabled a solution to dsDNA donor toxicity, but still require long homology arms and the need for AAV packaging, which can be limiting for fast-paced CRISPR research applications in many labs. Prime Editing enables delivery of the Cas9 protein and pegRNA as a purified complex, avoiding dsDNA toxicity and the need for viral packaging.

Standing on the Shoulders of Nature

The new editing mechanism that Prime Editing enables for CRISPR has an ancient evolutionary precedent in nature called target-primed reverse transcription (TPRT), first characterized in detail by Alan Lambowitz and team at the University of Texas, Austin (Zimmerly et al., 1995). The TPRT mechanism was illuminated by studying how selfish genetic elements, called group II introns, spread themselves throughout the bacterial world. During their studies of TPRT, the Lambowitz lab found that group II introns use a single protein (LtrA) and an intron RNA to target specific genomic sequences an insert copies of themselves. They located regions of the intron RNA which were responsible for binding to genomic DNA, serving a guide RNA function similar to CRISPR’s mode of operation. In contrast to Prime Editing, when the LtrA proteins of group II introns reverse transcribe copies of the intron RNA into the genome, they copy a much larger fragment of 1,000 bp or larger. While applications of group II introns remain limited largely to bacteria, the TPRT mechanism allows them to transcend the DNA repair intricacies of various bacterial species and made TargeTron the first widely portable tool for microbial genome engineering.

Prime Editing with CRISPR has introduced an unnatural complex, Cas9 fused with a reverse transcriptase, that makes TPRT viable in mammalian systems for small edits (1 to about 40 bases). Similar to the group II introns in bacteria, Prime Editing allows the efficient replacement of stretches of mammalian genomic DNA with RNA-specified edits. Ultimately, new approaches like Prime Editing, which are based on the TPRT mechanism, are poised to make the next generation of genome engineering tools even more precise and applicable than the current systems.

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