Invention Title:

GENOME EDITING USING CAS9 NICKASES

Publication number:

US20260218241

Publication date:

2026-07-30

Section:

Chemistry; metallurgy

Class:

C12N15/907

Inventors:

Feng Zhang 🇺🇸 Cambridge, MA, United States

Fei RAN 🇺🇸 Cambridge, MA, United States

Assignees:

President and Fellows of Harvard College 🇺🇸 Cambridge, MA, United States

MASSACHUSETTS INSTITUTE OF TECHNOLOGY 🇺🇸 Cambridge, MA, United States

THE BROAD INSTITUTE, INC. 🇺🇸 Cambridge, MA, United States

Applicants:

Massachusetts Institute of Technology 🇺🇸 Cambridge, MA, United States

PRESIDENT AND FELLOWS OF HARVARD COLLEGE 🇺🇸 Cambridge, MA, United States

The Broad Institute, Inc. 🇺🇸 Cambridge, MA, United States

Smart overview of the Invention

The invention focuses on the delivery, engineering, and optimization of CRISPR-Cas9 systems for precise genome editing. It emphasizes the creation and use of vectors that encode components of the CRISPR complex, ensuring enhanced specificity and reduced toxicity in both prokaryotic and eukaryotic cells. The application details methods for directing CRISPR complex formation to improve the accuracy of target recognition.

Technical Background

Advancements in genome sequencing have highlighted the need for precise genome targeting technologies. While existing genome-editing tools like zinc fingers and TALEs exist, the CRISPR-Cas9 system offers a simpler, faster alternative. Unlike other systems, Cas9 uses a guide RNA for DNA targeting, making it easy to design and implement for various applications, including synthetic biology and gene therapy.

Cas9 Mechanism

The Cas9 enzyme, particularly from Streptococcus pyogenes, is widely used for genome editing due to its ability to induce double-strand breaks (DSBs) at specific sites. These breaks activate DNA repair pathways, allowing for precise genome modifications. However, off-target effects remain a concern, necessitating improved specificity through engineered Cas9 variants, such as nickases.

Nickase Innovation

Cas9 nickases, created by mutating catalytic residues, induce single-strand nicks instead of DSBs, promoting non-mutagenic repair pathways. Double-nicking strategies using two nickases can effectively create DSBs with reduced off-target effects. This approach maintains on-target efficiency while minimizing unwanted mutations, making it suitable for therapeutic applications.

Future Directions

Despite advancements, challenges remain in enhancing homology-directed repair (HDR) efficiency in genome editing. Continued research aims to refine these techniques, ensuring higher precision and efficiency for therapeutic and biotechnological applications. The CRISPR-Cas system's potential to simplify genetic mapping and disease research underscores its significance in modern biotechnology.