Pioneering genome editing method could correct genetic diseases

CRISPRs cure genetic disease

Date: 23rd October 2019

Scientists currently estimate that a shocking 10,000 human diseases are caused by errors or a single error in a single gene.  With this statistic not taking into consideration complex or multi-factorial disorders the total number of diseases caused by faulty genes is almost unfathomable.

With over 300 million people worldwide suffering with a gene disorder and with 95% lacking an FDA-approved treatment the hunt for effective therapies is at the forefront of healthcare.

One of the most promising gene replacement or editing techniques currently under intense scrutiny is CRISPR-Cas9 technology.  It has been hailed as a potential cancer cure, a HIV treatment and is in fact, is one of the few FDA-approved treatments for an inherited genetic blood disorder, β thalassemia.

Now scientists have created a Cas9-based system, called prime editing, which offers versatile and precise genome editing by directly writing new genetic information into specific DNA sites.

David Liu and his team from the Broad Institute of MIT and Harvard, US, have designed a ‘search-and-replace’ genome editing technology using a catalytically impaired Cas9 (nickase) tethered to an engineered reverse transcriptase (RT) dubbed the prime editor (PE).

In tandem they modified guide RNAs, which usually target the Cas9 to specific sites in the genome, to create prime editing guide RNA (pegRNA), which contains both a DNA-targeting and template repair domain. Together, the PE & pegRNA system, therefore, specify both targeting and encoding of the newly desired edit template.

The mechanism for this is simple. First the PE and pegRNA complex target the DNA site and create a single stranded ‘nick’.  The 3’ end of the pegRNA, then binds to the 3’end of the ‘nicked’ DNA, and primes reverse transcript synthesis by acting as the template to produce stably edited DNA.

prime editor repairs genome


Validation of the prime editing

Initial tests in vitro demonstrated successful DNA repair of reporter genes containing premature stop codons in yeast.

To develop the first system for mammalian cell gene editing, PE1, the researchers transfected human cells with two plasmids, one encoding the PE elements and the other the pegRNA.  After optimising the primer-binding site length and RT placement at the C-terminus of Cas9 nickase, they showed successful genome editing properties.  Testing on four additional genomic sites established the ability of PE1 to directly install a range of gene edits without requiring double stranded breaks or DNA templates.

Next, they hypothesised that engineering the system further might improve targeting and efficiencies. First they looked at how changing the RT might improve the efficiency of DNA synthesis during prime editing and a combination of mutations were incorporated to create PE2, which had 1.6- to 5.1-fold increased efficiencies in editing point mutations over PE1.

The optimal pegRNA structure was then established, and the inclusion of a simple second gRNA allowed the original nickase to subsequently nick the non-edited strand of DNA to increase prime editing efficiency further.  Care was taken to avoid locations that would create double stranded breaks and the prime editor 3 system came to life (PE3).

prime editor repairs genome


The paper displays an impressive range of tests showing the versatility of the system. More than 175 edits in human cells were performed including

  • All possible base-to-base conversions
  • 19 insertions from 1bp up to 44 bp
  • 23 deletions up to 80 bp
  • 119 point mutations including
  • 83 transversions
  • 18 combination edits at 12 endogenous loci in the human and mouse genomes
  • Insertion of tags (Flag, His)
  • Efficiently corrected the primary genetic causes of sickle cell disease (transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA)
  • Successful use in four human cell lines and primary post-mitotic mouse cortical neurons
  • Off-target effects were 4.4-fold lower than Cas9+sgRNAs

It is clear this is an important advancement in gene therapy and the authors estimate that, in principle, this system could correct about 89% of known pathogenic human genetic variants.

Prime editing future

As with all new methods additional research is needed to assess off-target effects, optimisation in other cell types and organisms and efficient delivery systems.

However, the versatility that this system has to offer is exciting and should bring about a wide range of applications that will advance our science from the lab to the clinic.

For those interested in delving further into this technology The Broad Institute has made the technology freely available to the academic and non-profit communities, with licensed use given to Prime Medicine and Beam Therapeutics.  For more info on the companies please explore our synthetic biology maps.

For a more in depth read please follow the link to The Broad Insitute’s news.

Anzalone, A. V., P. B. Randolph, J. R. Davis, A. A. Sousa, L. W. Koblan, J. M. Levy, P. J. Chen, C. Wilson, G. A. Newby, A. Raguram and D. R. Liu (2019). “Search-and-replace genome editing without double-strand breaks or donor DNA.” Nature.

Figure adapted from The Broad Institute