Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa)

Abstract

Alfalfa ( Medicago sativa ) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green ( MsSGR ) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

“…The allelic variants with SNPs in sgRNA2 target region displayed very few edits and contributed to the overall lower editing efficiency at this site. This suggests that the choice of gRNA and the combination of sgRNAs offers opportunities to tailor the desired co-editing efficiency in sugarcane, similar to what is reported for other polyploid crops (Andersson et al, 2017;Liu et al, 2018;Wolabu et al, 2020).…”

Multigene editing reveals that MtCEP1/2/12 redundantly control lateral root and nodule number in Medicago truncatula

“…Although CRISPR/Cas9 technology has been utilized for targeted mutagenesis in M. truncatula , the present system is not sufficient for multigene editing ( Meng et al , 2017 ; Wolabu et al , 2020 ). We first tested the original CRISPR/Cas9 toolkit for Arabidopsis ( Xing et al , 2014 ) with the target gene STF in M. truncatula R108, as the knockout mutation exhibits apparently visible phenotype changes in young seedlings ( Tadege et al , 2011 ).…”

“…For the legume model plant M. truncatula , CRISPR/Cas9 techniques have been broadly applied for single-target mutagenesis ( Meng et al , 2017 ). Recently, a new multiplex genome editing system in tetraploid alfalfa ( Medicago sativa ) based on a polycistronic tRNA – gRNA approach was created by Wolabu et al (2020) , but still uses the U6 promoter cloned from Arabidopsis and focuses on multiple sites of single gene editing. However, multigene editing remains difficult to perform, because few native MtU6 promoters, but not MtU6-1p , have been confirmed to be valid for adequately driving small guide RNA (sgRNA) cassette expression in M. truncatula , which is necessary for multigene targeting in this species.…”

Functional characterization of PETIOLULE‐LIKE PULVINUS (PLP) gene in abscission zone development in Medicago truncatula and its application to genetic improvement of alfalfa

“…These observations indicate that the existence of organ boundaries influences AZ formation. Several genes that are involved in organ boundary formation have been shown to be involved in AZ development including BOP1 / 2 , ATH1 , AS1 and HWS (Gómez‐Mena and Sablowski, 2008; González‐Carranza et al ., 2007; Gubert et al ., 2014; Hepworth et al ., 2005; Jun et al ., 2010; McKim et al ., 2008; Norberg et al ., 2005; Xu et al ., 2008). Our study provides more evidence to support the potential link between organ boundary formation and AZ development.…”