Considerations in adapting CRISPR/Cas9 in nongenetic model plant systems

Abstract

The rapid development and application of the CRISPR system in genome editing and other applications clearly illustrate its revolutionary role in biological research. Its massive impact is similar to that of molecular cloning and PCR technologies (Yin et al., 2017). The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) (see Box 1 for all abbreviations used in this article) system originated from the microbe immune system and was adapted to provide powerful tools to enable genome editing (Belhaj et al., 2015;Lander, 2016). The constantly expanding CRISPR toolbox comprises various Cas proteins (e.g., Cas9, Cas12a, and Cas13) and their engineered variants, as well as orthologs from diverse bacterial species (Zhang et al., 2019a). In addition to genome editing, CRISPR technology has also been widely applied in transcriptome regulation and epigenome editing (Zhang et al., 2019a). Among various CRISPR systems, engineered class 2 CRISPR/Cas9 is the most popular and robust, especially the Cas9 from Streptococcus pyogenes (SpCas9). Therefore, unless otherwise noted, this review is focused on studies of CRISPR/SpCas9, shortened as CRISPR/Cas9, in plant genome editing. There are two components of the CRISPR/Cas9 system: the Cas9 endonuclease and the single-guide RNA (sgRNA) (Fig. 1). The ribonucleoprotein Cas9-sgRNA complex recognizes and binds any genomic regions that contain a protospacer adjacent motif (PAM) sequence, which is NGG (where N represents any nucleotide) for SpCas9. If the spacer sequence of the sgRNA (i.e., the first 20 nucleotides at its 5′ end; Fig. 1) matches the genomic sequence immediately upstream of the PAM sequence, Cas9 will cleave both strands of the genomic DNA, leaving blunt ends at the position between the third and fourth nucleotides upstream of PAM (Fig. 1; Jinek et al., 2012). The double-stranded DNA break (DSB) will be repaired by one of the two innate DNA repair systems: the non-homologous end-joining (NHEJ) pathway or homology-directed repair (HDR) pathway (Symington and Gautier, 2011). The error-prone NHEJ pathway is efficient and could introduce a small insertion or deletion (indel) at the DSB point (Fig. 1). When occurring in a gene-coding region, the indel might lead to a frameshift mutation or a premature stop codon in the target gene, and this approach has been widely used for gene knockouts (reviewed in Karkute et al., 2017;Bewg et al., 2018;Modrzejewski et al., 2019;Zhang et al., 2019a). In addition, the occurrence of a CRISPR-mediated indel in the promoter of a gene might interfere with transcription factor binding and alter

Transcriptome Dynamics of the Inflorescence in Reciprocally Formed Allopolyploid Tragopogon miscellus (Asteraceae)

“…Our hypothesis requires further study — an effect of pectin metabolism on inflorescence development has not been reported in Asteraceae. With an established CRISPR/Cas9 system in Tragopogon ( Shan et al, 2018 , 2020 ), the functions of related genes (such as genes encoding pectinesterase and polygalacturonase) could be rigorously examined.…”

Efficient CRISPR/Cas9-Mediated Gene Editing in an Interspecific Hybrid Poplar With a Highly Heterozygous Genome

“…Between 2013 and 2019, CRISPR/Cas9 technology has been successfully used to modify plant species from 45 genera across 24 families ( Shan et al., 2020 ). However, the application of the CRISPR/Cas9 system to genetically alter woody perennials has been limited to only the following 10 species: apple ( Nishitani et al., 2016 ), cacao ( Fister et al., 2018 ), cassava ( Odipio et al., 2017 ), citrus ( Jia et al., 2017 ), coffee ( Breitler et al., 2018 ), grape ( Nakajima et al., 2017 ), kiwifruit ( Wang et al., 2018 ), Parasponia andersonii ( Van Zeijl et al., 2018 ), pomegranate ( Chang et al., 2019 ), and poplar ( Fan et al., 2015 ; Zhou et al., 2015 ).…”

Biotechnological Perspectives of Omics and Genetic Engineering Methods in Alfalfa

“…Protein synthesis and processing: peptidyl-prolyl cis-trans isomerases, protein disulfide isomerase-like protein precursor, porin, proteasome subunit β type, eukaryotic translation initiation factor 3 subunit I, BiP isoform A/glycine max, cysteine proteinase, outer plastidial membrane protein porin Intracellular traffic, cell structure: protein TOC75, translocon Tic40, profilin Nitrogen metabolism: L-asparaginase Auxin polar transport: stilbene synthase family protein, monothiol glutaredoxin-S17 protein Lignin biosynthesis: cinnamyl alcohol dehydrogenase Pyruvate metabolism and transport: pyruvate carrier protein Du et al (2018) Vitamin B1 metabolism: thiazole biosynthetic enzyme (TAL effector nucleases or TALENs), and clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9). Among these, TALENs and CRISPR/Cas9 are the preferred SSNs for research purposes (Kanaar et al, 1998;Pastwa and Blasiak, 2003;Smith et al, 2006;Pâques and Duchateau, 2007;Hartlerode and Scully, 2009;Sander et al, 2011;Qi, 2015;Steinert et al, 2016;Malzahn et al, 2017;Shan et al, 2020). The history of gene targeting technologies started in 1988 when the first gene-targeting experiment was performed on tobacco (Nicotiana tabacum) protoplasts (Paszkowski et al, 1988).…”

“…Subsequently, TALENs were introduced into the group of plant genome editing technologies (Christian et al, 2010). Finally, CRISPR/Cas9 technology has been used in plants such as Arabidopsis thaliana, N. benthamiana, Oryza sativa, and T. aestivum (Li J. F. et al, 2013;Nekrasov et al, 2013;Shan et al, 2013Shan et al, , 2020.…”

“…Utilization of CRISPR/Cas9-based mutagenesis in several non-leguminous plant species, including data on delivery method, integration into the genome, and editing efficiency, has been reviewed recently (Belhaj et al, 2013;Jaganathan et al, 2018;Kuluev et al, 2019;Mao et al, 2019; FIGURE 1 | Overview and integration of omics and molecular genetics approaches aiming to improve agronomic traits and performance of alfalfa. Moradpour and Abdulah, 2020;Shan et al, 2020). Approaches such as transgene integration and gene stacking developed for diploid crop species (e.g., corn, cotton, soybean) might be less suitable for alfalfa due to its auto-tetraploid character (Kumar et al, 2018), but the CRISPR/Cas9 technology seems to work well.…”