Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA

Abstract

An article published in Nature (Anzalone et al., 2019) reports the development of a genome editing experimental approach that mediates all possible base-to-base conversions, "indels, " and combinations in human genome without the need of double-strand breaks (DSBs) or donor DNA (dDNA) templates. Prime editing, the novel method of genome editing, exploits a longer-than-usual single guide RNA (gRNA), known as prime editing gRNA (pegRNA), and a fusion protein consisting of Cas9 H840A nickase fused to an engineered reverse transcriptase (RT) enzyme. Described as "search-and-replace" base-editing technology, prime editing supplies the desired genetic construct in an extension to the gRNA, which is then converted to DNA using the RT enzyme. The new approach eliminates the need for co-delivery of a corrective DNA template, performs all possible nucleotide substitutions (including those for a sizeable proportion of genetic disorders), resolves frameshifts induced by indels and confers fewer off-target edits when compared with conventional CRISPR-Cas devices. Prime editing is an exciting new complement to existing CRISPR editing systems and may even be an improvement in many cases. However, prime editing introduces new challenges. Overcoming these obstacles and applying prime editing in vivo, will give rise to new genome editing therapies for rare genetic diseases.

Recent Advances in Genome Editing Tools in Medical Mycology Research

“…Prime editing, which can be used in biological systems, first searches for the points in the gene that need to be modified, then impose the editing effects, without breaking into double stranded DNA at the target points, through base deletion, insertion, and substitutions. The most notable advantage of this method to CRISPR is that it has less off-target effects and is more precise [ 115 , 116 , 117 ]. This technique is still in its infancy and has not yet been used in pathogenic fungi.…”

Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

“…Further shortcomings of the system include the large size of the fusion protein and the potential for random incorporation of cDNAs by the RT [248]. More in-depth reviews on BEs and PEs can be found elsewhere [249][250][251][252][253][254][255].…”

Precise Genome Editing in Poultry and Its Application to Industries

“…Despite the advantages of prime-editing technology, the protein structure is currently too large for efficient delivery in vitro or in vivo, and it cannot be used to induce precise modifications in large indel mutations. Therefore, for this approach to be applied more broadly, further research is needed to develop more optimized systems that can overcome current limitations and increase efficiency and specificity [ 39 , 40 ].…”