Genome Editing in Soybean with CRISPR/Cas9

Liu, Junqi; Gunapati, Samatha; Mihelich, Nicole T.; Stec, Adrian O.; Michno, Jean Michel; Stupar, Robert M.

doi:10.1007/978-1-4939-8991-1_16

Cited by 29 publications

(15 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Soybean methods, already known, have been used for the edition by CRISPR including the construction of binary vectors with the insertion of T-DNA ( Chen et al, 2021 ), biolistic bombardment ( Sun et al, 2015 ), and electroporation treatments ( Liu et al, 2019a ). Here, we report for the first time that CRISPR/Cas9 was used in the DNA-free target mutagenesis of soybean material by RNP delivery into protoplasts without the application of any DNA vectors ( Figures 1 – 6 ).…”

Section: Discussionmentioning

confidence: 99%

A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery

et al. 2022

View full text Add to dashboard Cite

CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the constitutive pathogen response 5 (CPR5) gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targeted deep sequencing strategies. We were able to identify different mutation patterns within insertions and deletions (InDels) between + 5 nt and –30 bp and mutation frequency ranging from 4.2 to 18.1% in the GmCPR5 locus. Our results showed that DNA-free delivery of Cas9 complexes to protoplasts is a useful approach to perform early-stage genetic screens and anticipated analysis of Cas9 activity in soybeans.

show abstract

Section: Discussionmentioning

confidence: 99%

A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery

et al. 2022

View full text Add to dashboard Cite

show abstract

“…The final construct was transformed into the disarmed Agrobacterium rhizogenes strain 18r12 ( Veena and Taylor, 2007 ). Whole plant soybean transformation was performed in the genetic background of the cultivar “Bert” ( Orf and Kennedy, 1992 ), sub-line Bert-MN-01, using previously published methods ( Paz et al., 2006 ; Liu et al., 2019 ). CRISPR/Cas9 reagents for whole plant transformation were assembled as described in Curtin et al.…”

Section: Methodsmentioning

confidence: 99%

Similar Seed Composition Phenotypes Are Observed From CRISPR-Generated In-Frame and Knockout Alleles of a Soybean KASI Ortholog

et al. 2020

Self Cite

View full text Add to dashboard Cite

The β-ketoacyl-[acyl carrier protein] synthase 1 ( KASI ) gene has been shown in model plant systems to be critical for the conversion of sucrose to oil. A previous study characterized the morphological and seed composition phenotypes associated with a reciprocal chromosomal translocation that disrupted one of the KASI genes in soybean. The principle findings of this work included a wrinkled seed phenotype, an increase in seed sucrose, a decrease in seed oil, and a low frequency of transmission of the translocation. However, it remained unclear which, if any, of these phenotypes were directly caused by the loss of KASI gene function, as opposed to the chromosomal translocation or other associated factors. In this study, CRISPR/Cas9 mutagenesis was used to generate multiple knockout alleles for this gene, and also one in-frame allele. These soybean plants were evaluated for morphology, seed composition traits, and genetic transmission. Our results indicate that the CRISPR/Cas9 mutants exhibited the same phenotypes as the chromosomal translocation mutant, validating that the observed phenotypes are caused by the loss of gene function. Furthermore, the plants harboring homozygous in-frame mutations exhibited similar phenotypes compared to the plants harboring homozygous knockout mutations. This result indicates that the amino acids lost in the in-frame mutant are essential for proper gene function. In-frame edits for this gene may need to target less essential and/or evolutionarily conserved domains in order to generate novel seed composition phenotypes.

show abstract

“…Besides these studies, CRISPR/Cas9 was also used to improve the quantity and quality of storage proteins in soybean seeds by creating of mutagenesis of seed storage protein genes (Li et al, 2019a). The detailed protocol for applying CRISPR/Cas9 technology in soybean is summarized by Liu et al (2019) and Bao et al (2020).…”

Section: Applications Of Crispr/cas9 In Legume Cropsmentioning

confidence: 99%

Applications of CRISPR/Cas9-based genome editing in the plant biology

Zheng¹,

Li²,

Ye³

et al. 2021

Turk J Bot

View full text Add to dashboard Cite

Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) is an acquired immune system found in bacteria and archaea that can specifically silence or degrade a foreign single or double strand nucleic acid to protect it from infection. In recent years, the CRISPR/Cas9 system has rapidly been evolved into a genome editing technology, in which the Cas9 endonuclease can be targeted to specific DNA sequences by guide RNAs (gRNAs) that are easily programmable. Due to simplicity, specificity and high efficiency, CRISPR/Cas9 is gradually replacing other gene editing technologies and has been implemented in basic and applied plant sciences to boost yield, regulate metabolic process, and increase stress resistance in different varieties. In current review, we introduced its application scope in scientific research and practical application. We summarized the procedure of target plant generation by CRISPR/Cas9 method. We mainly reviewed the applications of CRISPR/Cas9 and its recent advances in model plants and other crop plants, attempting to provide a related general information to researchers. Further, we also included the inadequacies and concerns of CRISPR/Cas9 that have emerged so far.

show abstract

Genome Editing in Soybean with CRISPR/Cas9

Cited by 29 publications

References 20 publications

A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery

A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery

Similar Seed Composition Phenotypes Are Observed From CRISPR-Generated In-Frame and Knockout Alleles of a Soybean KASI Ortholog

Applications of CRISPR/Cas9-based genome editing in the plant biology

Contact Info

Product

Resources

About