Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

Author(s)

Basso, Marcos Fernando ^{[1, 2]} ; Duarte, Karoline Estefani ^{[1, 3]} ; Santiago, Thais Ribeiro ^{[1, 4]} ; de Souza, Wagner Rodrigo ^[3] ; Garcia, Bruno de Oliveira ^[1] ; Brito da Cunha, Barbara Dias ^[1] ; Kobayashi, Adilson Kenji ^[1] ; Correa Molinari, Hugo Bruno ^[1]

Affiliation

^[1] Brazilian Agr Res Corp EMBRAPA, Natl Ctr Agroenergy Res CNPAE, BR-70770901 Brasilia, DF - Brazil

^[2] Mato Grosso Cotton Inst IMAmt, BIOMOL BIOTEC Lab, BR-78740970 Rondonopolis, MT - Brazil

^[3] Fed Univ ABC UFABC, Ctr Nat & Human Sci, BR-09606045 Sao Bernardo Do Campo, SP - Brazil

^[4] Fed Univ Brasilia Brasilia UNB, Dept Phytopathol, BR-70910900 Brasilia, DF - Brazil

• Document type: Journal article
• Source: PLANT BIOTECHNOLOGY; v. 38, n. 2, p. 227-238, JUN 2021.
• Web of Science Citations: 0

Abstract

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes. (AU)

• FAPESP's process: 19/04878-7 - Biotechnological strategies for genetic improvement of grasses for biofuel production
• Grantee: Wagner Rodrigo de Souza
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Young Investigators Grants