Unpredicted central inversion in a sgRNA flanked by inverted repeats

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 technology relies on single-guide RNAs (sgRNAs) to direct gene editing.
• sgRNAs are typically cloned into plasmid vectors for use in gene editing applications.
• Understanding potential sequence alterations during cloning and transformation is crucial for reliable CRISPR experiments.

Purpose of the Study:

• To investigate an observed phenomenon of sgRNA sequence inversion during CRISPR/Cas9 gene knockout experiments.
• To identify the mechanism behind the sgRNA inversion.
• To provide recommendations for sgRNA design and experimental procedures to prevent such alterations.

Main Methods:

• Transformation of sgRNA-containing plasmids into Escherichia coli.
• Sequence analysis of sgRNAs post-transformation to detect alterations.
• Investigation of plasmid structure, including the presence of inverted repeats.
• Experimental manipulation (sealing nicks) to assess impact on inversion.

Main Results:

• A central portion of the sgRNA sequence was found to be inverted after transformation into E. coli.
• The inverted segment was flanked by inverted repeat sequences within the sgRNA.
• Sealing nicks in the plasmid DNA successfully corrected the sgRNA inversion.
• The observed sgRNA recombination resulted in a complete alteration of the original sgRNA sequence.

Conclusions:

• sgRNA sequences can undergo unintended inversion within Escherichia coli due to flanking inverted repeats.
• This sgRNA recombination alters the functional sequence, potentially leading to failed or altered gene editing outcomes.
• Careful consideration of sgRNA sequence structure and potential recombination events is necessary during the design and execution of CRISPR/Cas9 experiments.