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, 17 (10), 1971-1984

Assessment of Cas12a-mediated Gene Editing Efficiency in Plants

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Assessment of Cas12a-mediated Gene Editing Efficiency in Plants

Joan Miquel Bernabé-Orts et al. Plant Biotechnol J.

Abstract

The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA-guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB-assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target-dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on-target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a-free segregating T2 lines to assess possible off-target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of -10 to -2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off-target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.

Keywords: CRISPR/Cas12a; CRISPR/Cas9; GoldenBraid; off-target; plant gene editing.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cas12a gene editing module (CGEM) assembly and strategy of the crRNA expression. (a) Level 1 cloning of the crRNA TU through BsaI restriction–ligation reaction requires three elements: AtU6‐26:DR (GB1442, GB1443) and partially complementary oligos of the PS and HDV termination signal (GB1444). BsmBI‐mediated binary assembly into level >1 of the crRNA TU and the As/LbCas12a TU (GB1440, GB1441) to create the Cas12a gene editing module. (b) Schematic of the constructs used for the expression of a single crRNA and the final structure of the transcript. Efficiency comparison between HDV and U6 poly‐T approach expressed as the percentage of undigested band after EcoRI digestion. Statistical t‐test analysis showed difference for AsCas12a and LbCas12a (P‐value <0.05). Error bars represent SEM; = 3. (c) Representation of the construct used for the expression of multiple crRNAs in the same transcript, and table showing the comparison of the efficiencies between HDV and multiplexing.
Figure 2
Figure 2
Comparison of the RGEN at several loci of N. benthamiana. (a) Mutation frequency for individual loci targeted with the three RGENs and determined by T7E1 mismatch cleavage assay. The sequence for each locus is detailed on the right part of the figure, with the Cas12a PAM highlighted in pink, the PS of Cas12a in light blue, the Cas9 PAM in purple and the PS in dark green. Error bars indicate SEM; = 3. ND: not detected. (b) Summary of the mutated and nonmutated loci for each endonuclease. Mean indel frequencies ± SEM are shown.
Figure 3
Figure 3
The DR loop sequence influences Cas12a activity. (a) Structure of the AsCas12a (AsDR), LbCas12a (LbDR) and synthetically engineered DR (synDR). Highlighted in pink are the different nucleotides of the three structures. The varying nucleotides of the synDR loops are also indicated (5′‐UNN‐3′) for each target. (b) Mutagenesis efficiency results of the wtDRs and synDRs at TFL1 and FT targets. All the DR structures were evaluated for each endonuclease. Error bars represent SEM; n = 3. ND: not detected.
Figure 4
Figure 4
Mutagenesis data extracted from stable transformations of different model plants. (a) Generic GEM encompasses three TUs: kanamycin selection marker (NptII), the crRNA expression cassette and the RGEN TU. (b) Representation of the XT1 loci of N. benthamiana with the targeted sequence, and table showing TIDE data from the mutated lines. Marked lines (*) were not analysed with TIDE. (c) Genomic loci of the S. lycopersicum MYB12 targeted gene, with the three RGENs, and results of the TIDE analysis of the edited plants. The ‘overall efficiency’ represents the sum of all the individual mutations traced by TIDE. The ‘mutation efficiency’ specifies the type of mutations associated with its efficiency in brackets. Only mutations whose P‐value <0.001 have been considered in ‘mutation efficiency’.
Figure 5
Figure 5
LbCas12a is capable of inducing large deletions in A. thaliana. (a) CGEM used for A. thaliana stable transformation and locus chosen to study the ability of LbCas12a to produce large deletions, showing the two target sites on the UTRs flanking the PDS3 gene. Arrows indicate primers used for the amplifications. (b) Alignments of three different complete deletions found in the white spots of the T1 generation visualized with Benchling. The complete genes have been deleted and the UTRs joined. (c) Analysis of albino phenotype (pds mutants, white seedlings) segregation in T2 seedlings grown in MS plates. Segregation analysis of DsRED(+) and DsRED(−) seeds from the three lines (PDS‐1, PDS‐2 and PDS‐3) and PCR amplification of white or green DsRED(−) seedling pools from the three different lines. (d) TIDE mutation analysis of DsRED(−) and DsRED(+) from pools of green plants from PDS‐1, PDS‐2 and PDS‐3 lines. Both target 1 (T1) and target 2 (T2) were analysed. ‘Overall efficiency’ and ‘mutation efficiency’ mean the same as in Figure 4.
Figure 6
Figure 6
Meta‐analysis of N. benthamiana, S. lycopersicum and A. thaliana mutation events. Compilation of all the TIDE data produced in the laboratory for Cas12a and Cas9 from transient and stable expression experiments of three plant species (N. benthamiana, S. lycopersicum and A. thaliana) to compare mutagenesis profile of both endonucleases.

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