Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops

Abstract

Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.

Prime editing is achievable in the tetraploid potato, but needs improvement

“…Here, we show for the first time that PPE2 strategy developed for dicot species is able to edit an endogenous gene in the cultivated tetraploid potato. Compared to our previous work on cytosine base editing in potato and Arabidopsis (Bastet et al, 2019;Veillet et al, 2019b;Veillet et al, 2019a;Veillet et al, 2020), the efficiency of our PPE strategy appears to be limited, as already observed for cereals. Once improved, the prime editing technology, by largely expanding the range of targeted base substitutions, could make precision breeding amenable in polyploid and vegetatively propagated crops such as potato.…”

CRISPR-induced indels and base editing using the Staphylococcus aureus Cas9 in potato

“…The PCR fragment was cloned into an intermediate pTwist plasmid through Mlu I/ Eco RI restriction followed by T4 DNA ligation (ThermoFisher Scientific, USA). A sequence encoding the PmCDA1 and UGI catalytic domains was previously dicot-codon optimized and synthesized (TwistBioscience, USA) [ 21 ], and cloned into the intermediate pTwist plasmid through EcoR I restriction and T4 DNA ligation (ThermoFisher Scientific, USA), downstream of the SanCas9 coding sequence. The construct was checked by sanger sequencing ( S1 Table ).…”

“…We first introduced a point mutation in the SaCas9 sequence to produce a SanCas9 (D10A) that we fused to a dicot codon-optimized fragment consisting in a cytosine deaminase (PmCDA1) and an uracil glycosylase inhibitor (UGI) domain. This fusion protein was cloned into a modified version of the pDe backbone [21,27,28], resulting in the pDeSanCas9_PmCDA1_UGI binary vector for expression in dicot species (Fig 3A). The four sgRNAs used for CRISPR-mediated indels were individually cloned into this CBE through Gateway cloning (Fig 3B), each spacer harboring two to five cytosines in the putative editing window (distal part of the spacer sequence from the PAM) for this CBE, based on previous studies using the PmCDA1 enzyme in plants with SpnCas9 or SanCas9 [15,19,21,29,30] ( Fig 3B).…”

“…These achievements allowed for the production of plants with new agronomic traits using gene knockout and/or base editing approaches, such as the production of tubers with low levels of amylose [ 17 – 19 ] or with improved resistance to harvest and post-harvest processes [ 20 ]. However, all the studies carried out so far on potato have used the native or engineered variants [ 21 ] of SpCas9, emphasizing the need to expand the CRISPR toolbox for this species which constitutes one of the most important crops for food production worldwide. In this study, we report on the successful use of the SaCas9 enzyme for both knockout and base editing applications in the tetraploid potato, confirming that the CRISPR-SaCas9 system constitutes a relevant alternative to the classical CRISPR-SpCas9 technology for functional studies and plant breeding.…”

Base Editing: The Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants

“…The xCas9 variant of SpCas9 could target NG, GAA, and GAT PAMs, which successfully expanded the scope of genome editing in rice [59,60]. In plants and human cells, SpCas9-NG variant was successfully tested that recognizes the NGN PAM [61][62][63][64]. Veillet et al [63] compared the efficiency of xCas9, SpCas9, and SpCas9-NG and found SpCas9-NG targets more efficiently alternative NGT PAMs in plants.…”