New tiny and precise CRISPR-Cas9 could bolster gene therapy techniques

Written by Alexander Marshall

A new, smaller CRISPR-Cas9 variant, which maintains precision, has been produced and could allow more effective gene therapy.

Researchers from City University of Hong Kong (Kowloon, Hong Kong) have created a new CRISPR protein. The new protein, termed SaCas9-HF, is designed to be smaller than the standard Cas9 protein while maintaining the high level of precision. Cas9’s size has been an obstacle to transitioning the technique into therapy so the researchers’ hope is that this new protein will allow the advance of clinical therapies.

Two major variants of the Cas9 protein have been derived from bacterial species, specializing in either precision, SpCas9, or a reduced size, SaCas9. While the larger protein with increased precision would be ideal for gene therapy, the size prevents the incorporation into some of the most accessible viral vectors, limiting its use in modifying human cells. In contrast, the SaCas9 can be easily incorporated but lacks the precision required for clinical therapies. In producing the new SaCas9-HF protein, the team hoped to maintain the level of precision seen from SpCas9 while enabling the Cas9 protein to be transmitted through adeno-associated virus vectors.

The research, led by Zongli Zheng and Jiahai Shi (both City University of Hong Kong), has demonstrated a 90% reduction in the number of off-target modifications compared with the standard SaCas9, while allowing the Cas9 to be transmitted by adeno-associated viruses. This would mean the Cas9 could be easily transferred and still produce precise modifications in human cells, creating a potential future in therapeutic use.

         “It will be particularly useful for future gene therapy using AAV vectors to deliver genome editing ‘drug’ in vivo and would be compatible with the latest ‘prime editing’ CRISPR platform, which can ‘search-and-replace’ the targeted genes,” claimed Zheng.

Adeno-associated viruses are rapidly becoming the predominant vector of choice for modification in human tissues due to their non-integrative, long-term potential; however, the inability to incorporate Cas9 has been a substantial obstacle to in vivo therapy. This new development may overcome that obstacle and whilst this is only one of many new advances in the use of CRISPR, Zheng suggests it may be incorporated into other systems to allow for more effective Cas9 delivery.

Sources: Y Tan, A Chu, S Bao et al. Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity. PNAS. 116(42) 20969-20976 (2019);

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What are adeno-associated viruses?

Adeno-associated viruses (or AAVs) are small viruses, which are utilized in gene therapy due to their small size, the minimal immune response, and ability to transport material into an array of cells.

Can CRISPR be used on adults?

Nothing limits the use of CRISPR on adult human cells in theory; however, the practicalities currently do not make this viable. The delivery methods which are available are often low efficiency or carry potential risks.

CRISPR has also, until recently, lacked the ability to produce the mutations patients would need to resolve a majority of genetic conditions, reducing its potential in a clinical setting.  

Where does CRISPR ‘cut’?

Utilizing simple-to-create guideRNAs, CRISPR is able to target a plethora of sites within the genome. The only large limitation is the presence of a proximal adjacent motif (PAM) site — an NGG sequence directly following the guide RNA sequence, three base pairs after the cut site.