Associate Director Science and Medicine, Yale University


Bill Hathaway

The promise of genome editing to help understand human diseases and create new therapies is vast, but technological limitations have limited advancement of the field. While existing editing technologies can alter or delete single base pairs within the human genome’s 3 billion base pairs, they are limited in their ability to alter multiple locations simultaneously — and can sometimes incorrectly alter neighboring DNA bases.

A new Yale study, however, advances the ability of scientists to edit multiple DNA sites by threefold and helps prevent unwanted mutations in nearby genetic sites.

The findings are published in the journal 

“We were able to increase the number of edits in a single cell while also enhancing the precision of these edits,” said Farren Isaacs, PhD, professor of molecular, cellular and developmental biology at Yale’s Faculty of Arts and Sciences and senior author of the study. Isaacs is also affiliated with the Systems Biology Institute at Yale West Campus and the Department of Biomedical Engineering at the Yale School of Engineering & Applied Science.

Advances in genome engineering have allowed researchers to more efficiently modify single genetic sequences that help improve the understanding of their biological roles. However, most human diseases, including cancer, arise from multiple genetic mutations.

“Many phenotypes arise from multiple genetic mutations but most gene editing has been focused on a single site, which has limited technology advancements in the field,” said first author Anabel Schweitzer, who is a student in Yale’s Graduate School of Arts and Sciences and a member of Isaacs’ lab.

Human genome editing historically has allowed for precise alterations of single base pairs by excising or inserting new sequences at a single location along the DNA strand. However, conventional gene editing technologies — such as CRISPR Cas9 — have been limited by the generation of double-strand breaks in DNA that introduce unwanted modifications in the genome. While the development of base editors has enabled direct chemical modification of target DNA nucleotides, enabling researchers to avoid DNA double-strand breaks, base editing technology has been constrained by the number and precision of single base edits that can be achieved.

If DNA is viewed as a massive 3 billion-character manuscript, the new engineering technology essentially allows researchers to make multiple changes in different chapters simultaneously, not just edit single words or sentences on one page. However, the improvements in genome engineering also have drawbacks. Sometimes edits are made at unintended locations which makes assessing the effects of the changes difficult.

For the new study, the Yale team used a CRISPR-associated protein Cas12 — which is similar to Cas9, a protein that can act as a sort of “molecular scissor” that can precisely cut or modify portions of DNA — and so-called guide RNAs (gRNAs). When fused to an enzyme, Cas9 and Cas12 can make targeted chemical changes to DNA at locations determined by the gRNA sequence. The team chose Cas12 because of its innate ability to process an RNA array containing many gRNAs. To improve the precision of editing, the team engineered the gRNAs by shortening the gRNA sequence or modifying the RNA bases.

They then used the new system to successfully alter gene sequences with greater precision at 15 different sites in human cells — three times as many locations as had been previously engineered.

The improvement will not only help assess the roots of complex genetic diseases, such as cancer, but will also guide the development of new designer drugs enabled by synthetic genomes developed in the Isaacs lab.

“Overcoming these key obstacles of mammalian genome engineering technologies will be critical for their use in studying single nucleotide variant-associated diseases and engineering synthetic mammalian genomes,” the authors say.

The research was primarily funded by Yale University and the National Institutes of Health.

Other Yale authors include Etowah Adams, Michael Nguyen, and Monkol Lek.

Articles

A recent study from Yale has significantly advanced genome editing by tripling the number of DNA sites that can be edited simultaneously, while reducing unwanted mutations. This breakthrough, achieved using CRISPR-associated protein Cas12 and engineered guide RNAs, enhances the precision of genetic modifications. The technology allows for multiple simultaneous edits, crucial for understanding complex diseases like cancer, which often involve multiple genetic mutations. This advancement not only aids in disease research but also paves the way for developing new therapies and synthetic genomes.

A new Yale study advances the ability of genome engineers to edit multiple DNA sites by threefold, and helps prevent unwanted mutations in the process.

