Precision correction of pathogenic mutations using prime editing technology

Author: Dr Christos Georgiadis, 18th December 2019


A broad range of genome editing tools are currently available enabling targeted scission at a predetermined DNA locus. The field has exploded with the invention of CRISPR allowing scientists to cheaply and rapidly screen reagents achieving highly efficient gene knockouts. Targeted knock-ins for the purpose of mutation correction have been on the contrary challenging inundated by reduced precision and excess byproduct formation. Anzalone et al. describe a novel technology, prime editing, which hopes to address these limitations and expand the scope of genome editing capabilities for the seamless correction of the vast majority of known human disease-causing genetic variants.


CRISPR has since its discovery in 2012 revolutionised the field of genome-editing enabling highly targeted and effortless modifications of the DNA. Facilitated by bacterial derived Streptococcus pyogenes Cas9 enzyme and guided by a short single guide RNA (sgRNA) sequence to a pre-determined genomic site the Cas9 nuclease creates a targeted double stranded break (DSB) in the DNA. Breaks can be resolved in one of two ways; most favourable is the non-homologous end joining (NHEJ) pathway resulting in rapid ligation of the broken ends which commonly leads to insertion/deletion (indel) formation and hence commonly used for gene knockout, alternatively, upon provision of an exogenous donor template sequence, the homologous directed repair (HDR) pathway may be triggered for purposes of gene addition or repair of a stretch of DNA.


However, the inherent nature of generating breaks and indels in the genome actively leads to DNA damage which may have unpredictable outcomes on cell fate. Moreover, the widely documented off-target activity associated with the spCas9 system has accelerated the search for a safer alternative.


One of these advancements published by the Liu group (Komor et al., 2016and Gaudelli et al., 2017) describes the fusion of rat-derived ‘apolipoprotein B mRNA editing enzyme, complex-1’ (APOBEC1) to a genetic variant of Cas9 modified to create single stranded nicks at the on-target DNA site. The fused APOBEC1 in turn chemically deaminates cytosine bases (C) to thymidine (T) within a 4 bp window of activity at the on-target DNA site. The technique known as base editing has been used successfully for the disruption of gene function as well as for the correction of pathogenic single nucleotide polymorphisms (SNPs), although this action remains restricted to C to T conversions and more recently A to G with adenine base editors (ABE). Moreover, deaminase-based editing is prone to modest bystander effects resulting in unwanted conversion of nearby bases reducing its precision as a mutation correction tool.


Prime editing forms the most recent innovation in the field building on the existing CRISPR platform and implementing clever molecular engineering allowing for high fidelity editing of a target site without the creation of DSBs. Elegantly described by Dr. Anzalone working in the Liu group at the Broad Institute in Cambridge, Massachusetts, this system fuses an RNA-programmable Cas9 to an engineered reverse transcriptase and a prime editing guide RNA (pegRNA) that serves both as a scaffold for the nuclease but also encodes an RNA template sequence that following its reverse transcription can insert new genetic information that replaces target DNA nucleotides. Referred to as a ‘search-and-replace’ technology it facilitates the creation of precise insertions, deletions, transitions, transversions, as well as a combinations of these changes greatly opening up the possibilities for correction of pathogenic mutations. With editing efficiencies reported to be between 20-50% this makes the platform comparable, and depending on the target, sometimes favourable to existing Cas9-initiated HDR approaches. Importantly, while indel formation of 1-10% still occurred with prime editing, off-target activity was lower than that of wild-type Cas9.


Proof of principle testing employed prime editing technology for the modelling and correction of mutations that cause Tay-Sachs disease and sickle cell anaemia. Prime editing was used first for the in vitro modelling of sickle cell disease, through incorporation of a single A•T to T•A mutation in the HBB gene in HEK293T cells. Clls homozygous for mutant HBB were selected and subsequently shown to be reverted to wild-type HBB with 58% efficiency with 1.4% indels. Similar modelling saw the installation of a known Tay-Sachs causing 4-bp insertion in HEXA using prime editing. The same technology was next employed for its correction through removal of the introduced insertion with 33% efficiency and 0.23% indel formation. Comparable results supporting prime editing were described for 175 separate edits tested in several cell lines as well as terminally differentiated mouse primary cells. The authors hope to next explore methods for improving delivery to a broad range of primary cell types while further testing its application in vivo. It will be of paramount importance, however, that additional research is carried out to fully elucidate the strengths and limitations of prime editing technology and characterise its off-target effects in a genome-wide manner.


It has been proposed that this technology has the potential to advance the correction of 89% of known pathogenic human genetic variants. While this novel technology is still in its infancy, it forms a noteworthy addition to the Cas9 toolbox and is expected to significantly expand the scope of genome editing.


References

Anzalone, A. V., et al. (2019). “Search-and-replace genome editing without double-strand breaks or donor DNA.” Nature 576(7785): 149-157.

Gaudelli, N. M., et al. (2017). “Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage.” Nature 551(7681): 464-471.

Komor, A. C., et al. (2016). “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533(7603): 420-424.