Journal of plant researchNov 16, 2024

Creating partly and completely disabled plants by using CRISPR to target mRNA splicing sites

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Apr 2, 2026

Abstract

Three mutant alleles of the HPY2 gene were generated, including a hypomorphic mutant that survived to the reproductive stage.

The hypomorphic mutant hpy2-cr3 has a five base pair deletion at an intron-exon junction, resulting in longer root growth compared to severe mutants.
Severe loss-of-function HPY2 mutants display dwarfism and seedling lethality, complicating functional analysis.
The hpy2-cr1 and hpy2-cr2 mutants, which have a single base pair insertion, exhibited significant root shortening and seedling lethality similar to the severe hpy2-1 allele.
RT-PCR analysis of hpy2-cr3 indicated the production of atypical mRNAs that generate truncated proteins, which retain some HPY2 function.
The generation of these novel hypomorphic mutants may facilitate the study of lethal HPY2 gene functions and complex genetic interactions.

Simplified

Genetic analysis is important for modern plant molecular biology, and in this regard, the existence of specific mutants is crucial. While genome editing technologies, particularly CRISPR-Cas9, have revolutionized plant molecular biology by enabling precise gene disruption, knockout methods are ineffective for lethal genes, necessitating alternatives like gene knockdown. This study demonstrates the practical generation of a hypomorphic mutant allele, alongside severe null mutant alleles, via the targeting of mRNA splicing sites using CRISPR-Cas9. The Arabidopsis HIGH PLOIDY 2 (HPY2) encodes a yeast NSE2 ortholog, part of the conserved eukaryotic SMC5/6 complex, with SUMO E3 ligase activity essential for cell cycle progression and plant development. Loss-of-function HPY2 mutants exhibit severe dwarfism and seedling lethality, making functional analysis challenging. To overcome these limitations, we created HPY2 knockdown mutants as novel tools to investigate gene function. Of the three mutant alleles, the hpy2-cr1 and hpy2-cr2 mutants resembled the existing severe hpy2-1 allele, both harboring a single base pair insertion in one exon, causing significant root shortening and seedling lethality. In contrast, the hypomorphic mutant hpy2-cr3, which has a five bp deletion at an intron-exon junction, showed relatively longer root growth and survived until the reproductive stage. RT-PCR analysis of hpy2-cr3 revealed atypical mRNAs producing truncated polypeptides that retained some HPY2 function, explaining the milder phenotype. These results establish the successful generation of novel hypomorphic mutant alleles critical for studying the lethal gene HPY2, and demonstrate the usefulness of CRISPR-Cas9 for producing viable hypomorphic mutants for investigating complex genetic interactions.

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Creating partly and completely disabled plants by using CRISPR to target mRNA splicing sites

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