Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• CRISPR-Cas9 and ssODNs enable targeted genomic alterations in iPSCs.
• Low ssODN knockin frequency limits precise genome editing.
• The biochemical behavior of Cas9 in mammalian cells remains underexplored.

Purpose of the Study:

• Investigate intrinsic cellular factors affecting Cas9 cleavage activity.
• Identify methods to enhance CRISPR-Cas9 genome editing efficiency.
• Optimize conditions for high-frequency ssODN knockin and allele replacement.

Main Methods:

• In vitro assays to assess Cas9 cleavage activity.
• Analysis of intracellular RNA, DNA, and protein fractions.
• Precomplexing Cas9 and sgRNA into ribonucleoprotein (RNP) complexes.
• Optimizing electroporation parameters for RNP and ssODN delivery.
• Generating custom-edited iPSCs by replacing HLA alleles.

Main Results:

• Intracellular RNA inhibits Cas9 binding to sgRNA and reduces enzymatic activity.
• Precomplexing Cas9-sgRNA (RNP) before cell delivery enhances genome editing compared to overexpression.
• Optimized electroporation achieved up to 70% single-nucleotide correction and 40% loxP insertion.
• Successful replacement of HLA-C1 with HLA-C2 alleles in iPSCs.

Conclusions:

• Intracellular RNA is a key inhibitor of Cas9 activity.
• Cas9-sgRNA RNP complex formation and optimized delivery are crucial for efficient genome editing.
• This optimized method significantly improves precise genome engineering in iPSCs, including allele replacement.