Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 and CRISPR-Cpf1 nucleases are widely used for mammalian genome editing.
• These nucleases introduce double-strand breaks repaired by homology-directed repair (HDR) with DNA donors.
• Improving the efficiency of precise HDR-mediated editing remains a key challenge.

Purpose of the Study:

• To develop a method to enhance precise genome editing efficiency.
• To evaluate the efficacy of a small molecule mixture, termed "CRISPY" mix, in conjunction with CRISPR/Cas9n and CRISPR/Cpf1.

Main Methods:

• Utilized CRISPR/Cas9n and CRISPR/Cpf1 nucleases for genome editing.
• Applied a novel mixture of small molecules (CRISPY mix) to enhance editing.
• Assessed editing efficiency in human induced pluripotent stem cells (hiPSCs) and other cell lines.

Main Results:

• The CRISPY mix increased precise genome editing with Cas9n by 2.8- to 7.2-fold, achieving ~50% chromosome editing for nucleotide substitutions and 27% for gene knock-in in hiPSCs.
• Editing efficiency with Cpf1 was improved 2.3- to 4.0-fold, enabling editing in ~20% of chromosomes.
• Observed cell-type specific variations in editing efficiency enhancement, suggesting dependence on cellular repair pathways.

Conclusions:

• The CRISPY mix significantly enhances precise genome editing efficiency with both Cas9n and Cpf1 nucleases, particularly in hiPSCs.
• Achieved record editing efficiencies in human stem cells, facilitating more effective genome engineering.
• The effectiveness of the CRISPY mix is influenced by cell type, highlighting the role of endogenous repair mechanisms.