Modified Protein Enhances the Accuracy of CRISPR Gene Therapy

A new protein that can enhance the accuracy of CRISPR gene therapy was recently developed by researchers from City University of Hong Kong (CityU) and Karolinska Institutet. This work, published in the Proceedings of the National Academy of Sciences, could potentially have a strong impact on how gene therapies are administered in the future.

CRISPR-Cas9 Gene Therapy

CRISPR-Cas9, often referred to as just CRISPR, is a powerful gene-editing technology that has the potential to treat a myriad of genetic diseases such as beta-thalassemia and sickle cell anemia. As opposed to traditional gene therapies, which involve the introduction of healthy copies of a gene to a patient, CRISPR repairs the genetic mutation underlying a disease to restore function.

CRISPR-Cas9 was discovered in the bacterial immune system, where it is used to defend against and deactivate invading viral DNA. Cas9 is an endonuclease, or an enzyme that can selectively cut DNA. The Cas9 enzyme is complexed with a guide RNA molecule to form what is known as CRISPR-Cas9. Cas9 is often referred to as the “molecular scissors,” being that they cut and remove defective portions of DNA. Being that it is not perfectly precise, the enzyme will sometimes make unintended cuts in the DNA that can cause serious consequences. For this reason, enhancing the precision of the CRISPR-Cas9 system is of paramount importance.

Two versions of Cas9 are currently being used in CRISPR therapies: SpCas9 (derived from the bacteria Streptococcus pyogenes) and SaCas9 (derived from Staphylococcus aureus). Researchers have engineered variants of the SpCas9 enzyme to improve its precision, but these variants are too large to fit into the adeno-associated viral (AAV) vector that is often used to administer CRISPR to living organisms. SaCas9, however, is a much smaller protein that can easily fit into AAV vectors to deliver gene therapy in vivo. Being that no SaCas9 variants with enhanced precision are currently available, these CityU researchers aimed to identify a viable variant.

Creating a More Precise Cas9 Variant

This recent research led to the successful engineering of SaCas9-HF, a Cas9 variant with high accuracy in genome-wide targeting in human cells and preserved efficiency. This work was led by Dr. Zheng Zongli, Assistant Professor of Department of Biomedical Sciences at CityU and the Ming Wai Lau Centre for Reparative Medicine of Karolinska Institutet in Hong Kong, and Dr. Shi Jiahai, Assistant Professor of Department of Biomedical Sciences at CityU.

Their work was based on a rigorous evaluation of 24 targeted human genetic locations which compared the wild-type SaCas9 to the SaCas9-HF. The new Cas9 variant was found to reduce the off-target activity by about 90% for targets with very similar sequences that are prone to errors by the wild-type enzyme. For targets that pose less of a challenge to the wild-type enzyme, SaCas9-HF made almost no detectable errors.

“Our development of this new SaCas9 provides an alternative to the wild-type Cas9 toolbox, where highly precise genome editing is needed,” explained Zheng. “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.”

Author Affiliations

Dr. Shi and Dr. Zheng are the corresponding authors of this publication. The first authors are PhD student Tan Yuanyan from CityU’s Department of Biomedical Sciences and Senior Research Assistant Dr. Athena H. Y. Chu from Ming Wai Lau Centre for Reparative Medicine (MWLC) at Karolinska Institutet in Hong Kong. Other members of the research team were CityU’s Dr. Xiong Wenjun, Assistant Professor of Department of Biomedical Sciences, research assistant Bao Siyu (now at MWLC), PhD students Hoang Anh Duc and Firaol Tamiru Kebede, and Professor Ji Mingfang from the Zhongshan People’s Hospital.