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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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Improved gene correction efficiency with a tailed duplex DNA fragment.

Hiroyuki Tsuchiya¹, Masayuki Uchiyama, Kazuhiro Hara

¹Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.

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Single-stranded DNA fragments, when modified into tailed duplexes, significantly improve gene correction efficiency compared to conventional PCR fragments. This method enhances gene editing for Hyg-EGFP and rpsL genes, offering a novel approach for genetic engineering.

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Area of Science:

Molecular Biology
Genetic Engineering
Biotechnology

Background:

Conventional Polymerase Chain Reaction (PCR) fragments are typically used for gene correction.
Single-stranded (ss) DNA fragments offer an alternative substrate for genetic manipulation.
Homologous recombination, mediated by proteins like RAD51, is a key pathway for DNA repair and gene correction.

Purpose of the Study:

To evaluate the efficiency of ss DNA fragments in gene correction compared to PCR fragments.
To investigate the role of oligonucleotide annealing in enhancing ss DNA-mediated gene correction.
To determine the impact of oligonucleotide properties on gene correction efficacy.

Main Methods:

Preparation of a 606-base ss DNA fragment via restriction enzyme digestion.
Annealing oligonucleotides to the ss DNA fragment to create a tailed duplex.
Assessing gene correction efficiency for a hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene.
Testing the method on another target gene, the rpsL gene.

Main Results:

ss DNA fragments, particularly as tailed duplexes, corrected the Hyg-EGFP gene more efficiently than PCR fragments.
Annealing oligonucleotides to the ss DNA fragment enhanced gene correction, especially when annealed at the 3'-region.
Oligonucleotide length and backbone structure influenced gene correction efficiency.
The tailed duplex method proved effective for correcting the rpsL gene as well.

Conclusions:

Tailed duplex DNA fragments are highly effective substrates for gene correction.
This method offers an improved alternative to conventional PCR fragments for genetic engineering.
The RAD51 protein may play a role in the enhanced gene correction observed with tailed duplexes.