Masters Research Rotation Submission

Daniel Moon (#3292)
Addressing the challenges of CRISPR/Cas9 development

GenKore:
GenKOre is a South Korea based company that provides gene therapy products and various gene editing-based solutions based on its own genetic scissors technology.

Genetic scissors technology induces changes in the sequence of a desired gene by defecting a guide module with a positioning function and a protein capable of modifying DNA.

This technology first developed into 1st generation zinc finger nuclease, then 2nd generation TALEN, and now 3rd generation CRISPR technology. GenKOre has secured UGEt technology that is differentiated from existing genetic scissors.

GenKOre has secured high-efficiency CRISPR-Cas12a technology. Cas12a, also known as Cpf1, has a relatively small size and less off-target effect than cas9, so it has strengths suitable for gene therapy, but has a disadvantage of relatively low editing efficiency.

In order to increase the efficiency of Cas12a, GenKOre added a UUUUAUUUU sequence to the 3' end of the guide RNA containing 20 nucleotides that bind to the target gene, resulting in an average two-fold improvement in efficiency compared to the existing efficiency.

Mammoth Biosciences:
Mammoth seeks therapeutic cures through CRISPR-based precision editing, leveraging novel CRISPR-Cas enzymes from their protein discovery platform.

They highlight ultra-small and flexible delivery with expanded delivery options. The small sizes of their Cas14 and CasΦ nucleases overcome the limitations to viral and non-viral delivery methods experienced with Cas9.

With alternative and shorter PAM requirements, newly discovered nucleases can direct edits to an expanded set of DNA sequences.

Scribe Therapeutics:
Scribe is building and applying a suite of CRISPR technologies into a single continually evolving and expanding genetic modification platform.

They are rapidly generating hundreds of synthetic CRISPR molecules and novel technologies every month and folding the best into a fully integrated set of genome editing modalities.

Scribe’s X-editing (XE) technology is their first engineered molecule built holistically on a novel CRISPR foundation to provide 3 key attributes needed for therapeutic use and in vivo genetic modification: greater activity, specificity, and deliverability.

Arbor Biotechnologies:
Arbor’s approach is one where they uncover novel gene editors and develop them into powerful tools in their quest for curative treatments for previously incurable genetic diseases.

Their portfolio consists of tailored nuclease therapies along with next generation editing capabilities to replace whole genes and to precisely correct mutations.

GenKOre’s competitive advantages:
Cas9 boasts efficacy but its larger size has proven to be a deterrent in delivery. In comparison, Cas12f is small enough to be delivered in a single AAV while also boasting efficacy thanks to the gRNA engineering.

GenKOre and Mammoth Biosciences were able to reduce the size but GenKOre still comes out ahead as Mammoth falls behind in efficacy. Scribe Therapeutics and Arbor Biotechnologies instead increased efficacy in other areas.

GenKOre’s Nature Biotechnology Article (September, 2021):
Cas12f1 has an extra-long gRNA for its compact protein size, which might be related to Cas12f1’s ssDNA cleavage activity. GenKOre’s engineered sgRNA_ge4.1, although still a bit longer, was structurally similar to the crRNA used by Cas12a or Cas12j.

The utility of CRISPR technology has been enormously expanded by engineering catalytic variants of Cas proteins and fusion with other functional proteins. Catalytically inactive Cas proteins have been fused to transcriptional regulators, thereby achieving tailored gene expression.

In particular, prime editing systems rely on a fusion of nCas9 and M-MLV reverse transcriptase for DNA modifications. Despite their functionalities, clinical applications of base and prime editors are limited by their weight beyond the limits of the genetic payload in an AAV.

Compact Cas proteins, such as Cas12f, should enable the creation of precise genome-editing tools that are easier to deliver with AAVs.

Cas12f is particularly efficient in the deletion of a pathogenic exon or intron using paired gRNAs. Usher’s syndrome, Duchenne muscular dystrophy, and a certain type of LCA could potentially be treated by such a Cas12f-based strategy.

To broaden the range of treatable diseases, it will be important to engineer catalytic variants of Cas12f1, which will benefit from the recent structural elucidation of Cas12f1, as witnessed for SpCas9 and Cas12a.