Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases
Vikash P Chauhan1,2, Phillip A Sharp1,2, Robert Langer1,3
1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.
Abstract:
CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications.
Insights
Researchers engineered a CRISPR-Cas9 variant (vCas9) to precisely control DNA repair pathways. This breakthrough enhances precise gene editing (HDR/MMEJ) while minimizing unwanted mutations (indels) for versatile applications.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Biotechnology
Background:
- CRISPR-Cas9 technology introduces targeted DNA breaks, leading to diverse mutations via competing repair pathways like nonhomologous end-joining (NHEJ) and homology-directed repair (HDR).
- Current control over these repair pathways is limited, influenced mainly by genomic context and cell state, restricting precise editing outcomes.
Purpose of the Study:
- To investigate engineered Cas9 nucleases that generate distinct DNA break structures to modulate competing DNA repair pathway frequencies.
- To develop a novel Cas9 variant (vCas9) capable of suppressing NHEJ and promoting precise repair pathways (MMEJ and HDR).
Main Methods:
- Engineering Cas9 variants to alter DNA break structures.
- Assessing the impact of different break structures on DNA repair pathway utilization (NHEJ, MMEJ, HDR).
- Evaluating the efficiency of vCas9 in precise editing and indel suppression across different cell types.
Main Results:
- Engineered Cas9 nucleases with altered break structures significantly changed repair pathway engagement frequencies.
- The developed vCas9 variant preferentially directs DNA breaks towards MMEJ and HDR pathways, suppressing dominant NHEJ.
- vCas9 demonstrated efficient precise editing via HDR or MMEJ and reduced indels in both dividing and nondividing cells.
Conclusions:
- DNA break structure is a critical determinant of repair pathway choice in CRISPR-Cas9 editing.
- vCas9 represents a significant advancement, enabling precise gene editing with suppressed unwanted mutations.
- This work establishes a new paradigm for designing targeted nucleases tailored for specific gene editing applications.
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