Targeting TOR and SnRK1 Genes in Rice with CRISPR/Cas9.

Area of Science:

• Plant Molecular Biology
• Genome Editing
• Agricultural Science

Background:

• CRISPR/Cas9 is widely used for gene mutation and knockout.
• Information on essential gene mutagenesis using CRISPR/Cas9 is limited.
• Essential genes OsTOR and OsSnRK1α in rice are crucial for plant development.

Purpose of the Study:

• To investigate the efficiency of CRISPR/Cas9 in targeting essential rice genes OsTOR and OsSnRK1α paralogs.
• To analyze the impact of mutations on plant development and identify loss-of-function phenotypes.

Main Methods:

• Constitutively expressed and inducible CRISPR/Cas9 systems were employed.
• Targeted mutagenesis of the singleton OsTOR and OsSnRK1α paralogs (OsSnRK1αA, OsSnRK1αB, OsSnRK1αC) in rice.
• Analysis of mutation rates, mutation types, and resulting phenotypes in primary mutants and surviving lines.

Main Results:

• Constitutive CRISPR/Cas9 successfully generated mutations in OsTOR and OsSnRK1α genes, with higher rates for OsSnRK1α paralogs.
• Inducible CRISPR/Cas9 did not yield detectable mutations.
• OsSnRK1α paralogs OsSnRK1αB and OsSnRK1αC were simultaneously targeted, generating double mutants; over 50% of primary mutants exhibited sterility or early senescence.

Conclusions:

• CRISPR/Cas9, particularly the constitutive system, is effective for mutating essential rice genes OsTOR and OsSnRK1α.
• Mutations in these essential genes lead to significant developmental defects, including sterility and senescence.
• Recovered lines display loss-of-function phenotypes, facilitating research into the roles of OsTOR and OsSnRK1α in rice development and environmental responses.