Heat-shock-inducible CRISPR/Cas9 system generates heritable mutations in rice

Area of Science:

• Plant biotechnology
• Molecular biology
• Genome editing

Background:

• CRISPR/Cas9 technology offers precise genome editing but requires controlled expression to limit off-target effects.
• Transient expression strategies are crucial for managing CRISPR/Cas9 activity and enhancing editing precision.

Purpose of the Study:

• To develop and evaluate a heat-shock (HS)-inducible CRISPR/Cas9 system for controlled genome editing in rice.
• To assess the efficiency, precision, and heritability of HS-induced targeted mutagenesis.

Main Methods:

• Constructed an HS-inducible CRISPR/Cas9 system in rice using soybean heat-shock protein gene and rice U3 promoters.
• Induced targeted mutagenesis at two loci in rice via heat shock treatment.
• Analyzed mutation rates, off-target mutations, and segregation in progeny using Sanger sequencing.

Main Results:

• Low basal mutagenesis (~16%) was observed without heat shock.
• Heat shock significantly increased targeted mutagenesis rates (50-63%) in transgenic rice lines.
• HS-induced mutations were efficiently transmitted to progeny and showed reduced off-target mutations compared to constitutive CRISPR/Cas9 systems.

Conclusions:

• The HS-inducible CRISPR/Cas9 system provides a controlled and efficient platform for genome editing in rice.
• This system effectively suppresses mutagenesis in the absence of heat shock and induces it upon treatment, improving editing precision.
• The technology holds promise for minimizing genome-wide off-target effects and advancing precise genome editing in crop improvement.