Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ...

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genome Editing

Background:

• CRISPR-mediated genome editing is crucial for genetic modification.
• Efficient, site-specific gene knock-in via homology-directed DNA repair (HDR) is challenging in plants, particularly woody species.
• Non-homologous end joining (NHEJ) often competes with HDR, reducing knock-in efficiency.

Purpose of the Study:

• To develop an improved CRISPR-mediated gene knock-in system in poplar.
• To enhance homology-directed DNA repair (HDR) efficiency by manipulating DNA repair pathways.
• To assess the impact of combined NHEJ inhibition and HDR enhancement on gene targeting and editing outcomes.

Main Methods:

• Simultaneous inhibition of non-homologous end joining (NHEJ) cofactor XRCC4 using silencing.
• Overexpression of HDR enhancer factors CtIP and MRE11.
• Integration of the BleoR gene into the 3' end of the MKK2 gene in poplar, with efficiency evaluated by TaqMan real-time PCR.

Main Results:

• HDR-mediated knock-in efficiency reached up to 48% with XRCC4 silencing and CtIP/MRE11 overexpression.
• This approach significantly improved genome targeting efficiency.
• CRISPR-induced insertions and deletions (InDels) were reduced 7-fold, with no observed functional effects on MKK2-based salt stress responses.

Conclusions:

• Simultaneous inhibition of NHEJ and overexpression of HDR enhancers significantly boosts CRISPR gene knock-in efficiency in poplar.
• This optimized system provides precise gene integration with minimal off-target mutations.
• The developed approach holds potential for improving gene editing in plants, crops, and potentially mammals.