# Novel methods to improve nuclease mediated homologous recombination > **NIH NIH R42** · ATGC, INC. · 2022 · $854,843 ## Abstract Project Summary The 2020 Nobel Prize in Chemistry was awarded to Drs. Emmanuelle Charpentier and Jennifer Doudna for their development of a revolutionary gene-editing tool, CRISPR/Cas9. It allows precise edits to the genome and has swept through the life science field. It has countless applications. Scientists hope to use it to develop therapeutic strategies for treating human genetic diseases. However, there are still several hurdles that need to be overcome before achieving clinical applications. One of the major concerns is the undesirable insertion or deletion (indel) events at off-target sites, as well as at the on-target site where the goal is to introduction precise correction or mutation. Another aspect that remains to be further improved is the low efficiency of knockin (KI) when a large size donor fragment is used, which is often below 1%. In Phase I of this STTR project, we engineered the spCas9 protein by fusing a 36 amino acid long peptide encoded by BRCA2 Exon 27 (Brex27), which has been reported to bind RAD51 to enhance homology-directed repair (HDR). We named this new variant the meticulous integration spCas9 (mi-spCas9), which possesses a unique combination of desirable features, including improving knock-in rates, reducing undesirable off-target events, and reducing undesirable on-target insertion or deletion (indel) events, providing a “one small stone for three birds” tool in gene editing. In this Phase II project, we propose studies to further engineer Brex27, to develop an Adeno Associated Virus (AAV) friendly mi-saCas9 and demonstrate its clinically relevant applications. Specifically, i) in Aim 1, we will develop next-generation mi-Cas9s (mi-spCas9-v2) towards near-complete abolishment of undesirable on-target and off-target indels; ii) in Aim 2, we will develop and optimize an AAV-friendly mi- saCas9 for in vivo gene editing; iii) in Aim 3, we will demonstrate the advantages of mi-Cas9s in clinically relevant applications. We expect that mi-spCas9-v2 and mi-saCas9 lead to a multi-fold increase in gene knock- in rates and close to zero on-target and off-target indel rates. Completion of the proposed studies will enhance the safety and efficacy of genome editing, propelling novel mi-Cas9 tools closer to an emerging multi-billion- dollar market of basic research and therapeutic. ## Key facts - **NIH application ID:** 10383251 - **Project number:** 2R42GM122181-02 - **Recipient organization:** ATGC, INC. - **Principal Investigator:** Jifeng Zhang - **Activity code:** R42 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2022 - **Award amount:** $854,843 - **Award type:** 2 - **Project period:** 2017-08-01 → 2024-03-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10383251 ## Citation > US National Institutes of Health, RePORTER application 10383251, Novel methods to improve nuclease mediated homologous recombination (2R42GM122181-02). Retrieved via AI Analytics 2026-06-03 from https://api.ai-analytics.org/grant/nih/10383251. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*