Utilizing CRISPR-Cas in Tropical Crop Improvement: A Decision Process for Fitting Genome Engineering to Your Species.

Kathleen A Joo,Michael B Kantar,Angel R Del Valle Echevarria,Michael G Muszynski,Ming-Li Wang,Xiaoling He

doi:10.3389/fgene.2021.786140

Kathleen A Joo, Michael B Kantar + Show 4 more

Open Access

https://doi.org/10.3389/fgene.2021.786140

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Abstract

Adopting modern gene-editing technologies for trait improvement in agriculture requires important workflow developments, yet these developments are not often discussed. Using tropical crop systems as a case study, we describe a workflow broken down into discrete processes with specific steps and decision points that allow for the practical application of the CRISPR-Cas gene editing platform in a crop of interest. While we present the steps of developing genome-edited plants as sequential, in practice parts can be done in parallel, which are discussed in this perspective. The main processes include 1) understanding the genetic basis of the trait along with having the crop’s genome sequence, 2) testing and optimization of the editing reagents, development of efficient 3) tissue culture and 4) transformation methods, and 5) screening methods to identify edited events with commercial potential. Our goal in this perspective is to help any lab that wishes to implement this powerful, easy-to-use tool in their pipeline, thus aiming to democratize the technology.

Highlights

Since its proposal as a eukaryotic gene-editing tool (Jinek et al, 2012), the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein (Cas) technology has been widely applied in microorganisms, animals, and plants to study gene function (Haque et al, 2018) due to its simplicity in design and straightforward execution
Sequence information about the gene or region in the chromosome associated with the trait of interest is critical in designing the appropriate gRNA respective to the Cas endonuclease (Figure 1B)
Having the genomic sequence allows for identification of the protospacer adjacent motif (PAM) sequence relevant to the Cas enzyme to be used (i.e., 5′-NGG-3′ for Cas9 (Jinek et al, 2012) or 5′-TTTV for Cas12a (Zetsche et al, 2015), where “N” is any nucleotide and “V” is A, G, or C) and subsequent gRNA design

Summary

INTRODUCTION

Since its proposal as a eukaryotic gene-editing tool (Jinek et al, 2012), the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein (Cas) technology has been widely applied in microorganisms, animals, and plants to study gene function (Haque et al, 2018) due to its simplicity in design and straightforward execution. The guides should be assessed for editing efficiency either in vitro or in vivo prior to tissue culture and transformation In both instances, the gRNA is incubated with their compatible Cas nuclease to form the RNP complex before testing (Woo et al, 2015; Li et al, 2016; Liang et al, 2017, 2018b; Banakar et al, 2019). A major challenge for CRISPR-Cas genome editing in tropical crops is often the lack of efficient tissue culture and transformation protocols due to their lengthy generation times (Haque et al, 2018; van Eck, 2018). We suggest reviewing Grohmann et al, 2019 for an overview of screening and selection methods to aid in protocol development

CONCLUSION

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