Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos

Area of Science:

• Genetics
• Molecular Biology
• Bioengineering

Background:

• Genome editing technologies like CRISPR-Cas9 offer potential for treating genetic diseases by correcting pathogenic mutations.
• Accurate assessment of off-target mutations is crucial for the safety and efficacy of genome editing applications.
• Individual genetic variations, such as single-nucleotide polymorphisms (SNPs), complicate the identification of true off-target editing events.

Purpose of the Study:

• To develop and validate a sensitive method for detecting genome-wide off-target mutations induced by genome editing tools.
• To compare the fidelity of different base editors and CRISPR-Cas9 in vivo.
• To establish a benchmark for off-target mutation rates in early mouse embryos.

Main Methods:

• Development of the Genome-Wide Off-target analysis by Two-cell embryo injection (GOTI) method.
• Editing of one blastomere in two-cell stage mouse embryos using CRISPR-Cas9 or adenine base editor (ABAE) or cytosine base editor (CBE).
• Whole-genome sequencing of progeny cells from edited and non-edited blastomeres at embryonic day 14.5 to identify off-target single-nucleotide variants (SNVs).

Main Results:

• GOTI successfully detected off-target mutations genome-wide.
• CRISPR-Cas9 and ABAE editing resulted in rare off-target SNVs, with frequencies comparable to spontaneous mutation rates.
• Cytosine base editors (CBEs) induced SNVs at frequencies over 20-fold higher than spontaneous rates, indicating lower fidelity.

Conclusions:

• GOTI is an effective method for assessing genome-wide off-target mutations.
• CRISPR-Cas9 and ABAE demonstrate high specificity for in vivo genome editing in early embryos.
• Cytosine base editors require further optimization to improve their fidelity and reduce unintended off-target mutations.