Efficient CRISPR/Cas9 Genome Editing in Alfalfa Using a Public Germplasm

Emilia Bottero,Gabriela Massa,Eduardo Blumwald,Hiromi Tajima,Gabriela Soto,Margarita Stritzler,Sergio Feingold,Matías González,Romina Frare,Cristina Gómez,Nicolás Ayub

doi:10.3389/fagro.2021.661526

Abstract

Because its ability to acquire large amounts of nitrogen by symbiosis, tetraploid alfalfa is the main source of vegetable proteins in meat and milk production systems in temperate regions. Alfalfa cultivation also adds fixed nitrogen to the soil, improving the production of non-legumes in crop rotation and reducing the use of nitrogen fertilizers derived from fossil fuel. Despite its economic and ecological relevance, alfalfa genetics remains poorly understood, limiting the development of public elite germplasm. In this brief article, we reported the high-efficiency of alfalfa mutagenesis by using the public clone C23 and the CRISPR/Cas9 system. Around half of the GUS overexpressing plants (35S-GUS under C23 genomic background) transformed with an editing plasmid containing two sgRNAs against the GUS gene and the Cas9 nuclease exhibited absence of GUS activity. Nucleotide analysis showed that the inactivation of GUS in CRISPR/Cas9-editing events were produced via different modifications in the GUS gene, including frameshift and non-sense mutations. Using the CRISPR/Cas9 system and two sgRNAs, we have also edited the alfalfa gene NOD26, generating plants with different doses of alleles at this locus, including complete gene knockout at high efficiency (11%). Finally, we discuss the potential applications of genome-editing technologies to polyploid research and to alfalfa improvement public programs.

Highlights

The production of mutant alleles has been essential for plant functional genetics
To evaluate the rate of induced mutation frequency via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in alfalfa by using the C23 genotype, we produced two transgenic alfalfa events (3-1 and 5-1) with the pBI121 binary vector containing the GUS gene (Figure 1A) and expressing high GUS activities in both somatic embryos and leaves (Figure 1B), which were re-transformed with the pGUS-MUT plasmid containing two single guide RNA (sgRNA) against the GUS gene under the control of the U6 promoter for the editing of this reporter via CRISPR/Cas9 (Figure 1A)
GUS inactivation was observed in plants derived from 3-1 to 5-1 transformed with the pGUS-MUT plasmid (Figure 1B), where the frequency of GUS inactivation was 48% (37 GUSnegative and 40 GUS-positive plants) and 62% (58 GUS-negative and 35 GUS-positive plants), respectively, showing the high efficiency of CRISPR/Cas9 genome editing system in C23 genomic background

Summary

INTRODUCTION

The production of mutant alleles has been essential for plant functional genetics. Efficient generation of stable plant mutant lines has been achieved using different methods, including ethyl methanesulfonate, ionizing radiation, RNA interference, artificial microRNAs, and the insertion of T-DNA and retrotransposons. There has been an explosion in the production of knockout plant lines through the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas genome-editing system of Streptococcus pyogenes, which requires the canonical PAM motif NGG (N, any nucleotide; G, guanine) in dsDNA-bound states for DNA targeting. This system contains two components: one encoding the Cas endonuclease and the other containing a synthetic single guide RNA (sgRNA), with the latter typically under the control of the U6 small nuclear RNA gene promoter. In this brief research report, we used this framework for the production of CRISPR/Cas gene-edited alfalfa from clone C23

MATERIALS AND METHODS

RESULTS AND DISCUSSION

DATA AVAILABILITY STATEMENT