Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes.

Area of Science:

• Plant biotechnology
• Molecular biology
• Genomics

Background:

• Accurate detection of genome editing-induced mutations is challenging in plants, particularly polyploids.
• Efficient screening methods are crucial for low-frequency mutations and large population analysis.

Purpose of the Study:

• To develop and validate a sensitive and widely applicable method for detecting genome editing mutations in plants.
• To assess the utility of the PCR/ribonucleoprotein (RNP) complex method in polyploid species and for various editing tools.

Main Methods:

• Utilized purified CRISPR ribonucleoprotein (RNP) complexes to cleave PCR products for mutation detection.
• Applied the method to hexaploid wheat and diploid rice, comparing its sensitivity and applicability to Sanger sequencing and PCR/restriction enzyme (RE) methods.
• Tested the detection of TALEN-induced mutations and base editing mutations using high-fidelity Cas9 variants.

Main Results:

• The PCR/RNP method demonstrated higher sensitivity than Sanger sequencing and broader applicability than PCR/RE methods.
• Successfully detected mutations in hexaploid wheat, overcoming challenges posed by surrounding single nucleotide polymorphisms.
• Enabled detection of foreign DNA-free TALEN-induced mutations and partial base editing mutations.

Conclusions:

• The PCR/RNP method offers a low-cost, highly applicable, and rapid solution for detecting genome-edited mutations in diverse plant species.
• This technique is particularly advantageous for genome editing in complex genomes like wheat and for screening large populations.
• The method supports the detection of various mutation types induced by different genome editing tools.