In this study, we show that exposing Arabidopsis or Citrus plants to repeated heat stress treatments has a major effect on the rate of mutagenesis by CRISPR/Cas9. Our work points to the Cas9 protein from Streptococcus pyogenes (SpCas9) as being less active at the normal temperatures used to grow plants. These results provide a new method to all researchers seeking to rapidly improve CRISPR/Cas9 efficiency in their experiments.

Aside from genome structure differences, another variable between different biological systems that could affect CRISPR/Cas9 activity is growth temperature. Many different biological parameters/processes are affected by temperature, and some of these (e.g. enzyme kinetics, chromatin structure, DNA repair pathways; Daniel et al ., 2001 ; Oei et al ., 2015 ; Pecinka et al ., 2010 ) could directly affect the ability of CRISPR/Cas9 to induce mutations in eukaryotic genomes. Therefore, it is possible that temperature could contribute to the observed variation in efficiency of CRISPR/Cas9 in different organisms, and that modulating growth temperature could significantly increase targeted mutagenesis.

In the model plant Arabidopsis thaliana , most of the work aiming to increase the rate of mutagenesis induced by CRISPR/Cas9 activity has focused on using different promoters to drive Cas9 expression (reviewed in Mao et al . ( 2017 )). Using different constitutive, tissue‐specific or germline‐specific promoters to express Cas9 has been shown to increase the frequency of mutations in first‐generation transgenic plants and the heritability of mutations in subsequent generations (Feng et al ., 2013 , 2014 ; Jiang et al ., 2013 ; Li et al ., 2013 ; Mao et al ., 2013 , 2016 ; Fauser et al ., 2014 ; Gao et al ., 2015 ; Hyun et al ., 2015 ; Wang et al ., 2015 ; Yan et al ., 2015 ; Tsutsui and Higashiyama, 2017 ). Another way to maximize Cas9 activity has been to use plant codon‐optimized versions of Cas9 (Bortesi and Fischer, 2015 ). Both promoter activity and codon‐optimized Cas9 increase targeted mutagenesis through the same mechanism, which is by increasing the levels of Cas9 proteins in plants.

CRISPR/Cas9 has become the leading tool in eukaryotes for targeted mutagenesis and precise engineering through homologous recombination‐based repair of double‐stranded DNA (dsDNA) breaks (Jinek et al ., 2012 ; Cong et al ., 2013 ). Genome editing using CRISPR/Cas9 has been shown to be possible in all biological systems, from bacteria to complex eukaryotes like animals and plants. However, variation in the efficiency of on‐target and off‐target mutagenesis between different organisms has been observed, which could be due to differences in genome structure (e.g. genome size, GC content) (Bortesi et al ., 2016 ). Much work remains to be done to optimize the bacterial‐derived CRISPR/Cas9 system for use in eukaryotes, which is required to maximize our ability to efficiently engineer different genomes.

Results

To assess the effect of temperature on targeted mutagenesis by CRISPR/Cas9, we first used Arabidopsis thaliana as a model system, as Arabidopsis is usually grown at 22°C but can sustain growth at much higher temperatures (Mittler et al., 2012). A previously described CRISPR/Cas9 system that consists of SpCas9 expressed under the Arabidopsis YAO promoter (pYAO::SpCas9) was used for this study (Yan et al., 2015). We also established an in vivo quantitative assay using a GFP reporter gene and flow cytometry to rapidly assess the rate of targeted mutagenesis at the cellular level in different tissues. In this assay, GFP is tagged to the C‐terminus of a histone H3.3 protein (HTR5) and expressed throughout the plant under the HTR5 promoter (pHTR5::HTR5‐GFP). We created T1 plants expressing pYAO::SpCas9 and a sgRNA targeting HTR5 in a homozygous background containing the pHTR5::HTR5‐GFP reporter gene. Therefore, the loss of GFP expression in these plant nuclei requires independent mutagenic events at two pHTR5::HTR5‐GFP loci, and potentially more in endoreduplicated cells, depending on the developmental timing of mutagenesis (Galbraith et al., 1991).

First‐generation transgenic (T1) Arabidopsis plants expressing pYAO::SpCas9, the sgRNA targeting HTR5 and pHTR5::HTR5‐GFP were either continuously grown at 22°C or exposed to four heat stress treatments at 37°C for 30 h until plants transitioned to reproductive growth (Figure 1(a)). Restricting exposure to heat stress at 37°C during the vegetative phase of growth prevents floral bud abortion later in development (Warner and Erwin, 2005). Plants were allowed to recover for 42 h at 22°C between heat stress treatments since continuous growth at 37°C was lethal in our growth conditions, while plants exposed repeatedly to short heat stress (SHS) treatments of 30 h displayed only a mild hyponastic leaf phenotype (Figure S1). As a control for the effect of SHS treatments on GFP expression/stability, we also exposed plants expressing pHTR5::HTR5‐GFP (but no SpCas9‐sgRNA) to heat stress, and did not observe any effect on GFP fluorescence (Figure 1(b)). We found that the percentage of GFP‐positive leaf nuclei was much lower in SHS plants (average of 12% for T1 plants) than in plants continuously grown at 22°C (average of 89% for T1 plants) (Figure 1(b)). We confirmed by sequencing that loss of GFP fluorescence is indicative of mutations at the HTR5‐GFP transgene, and observed that a single nucleotide insertion at the predicted cleavage site is the most common type of allele recovered in plants grown at 22°C or exposed to heat stress (Figure 1(c) and Figure S2). We repeated this experiment using nuclei derived from flowers instead of leaves, and observed a similar enhancement of targeted mutagenesis in plants exposed to multiple SHS (Figure 1(d)). All the plants used in these assays were from the same T1 population homozygous for pHTR5::HTR5‐GFP, which negates the possibility that differences in copy numbers of Cas9 or the reporter gene between stressed and unstressed plants account for the results obtained. Taken together, our results show that temperature is a critical parameter regulating targeted mutagenesis by CRISPR/Cas9 in somatic tissues of Arabidopsis.

Figure 1 Open in figure viewer PowerPoint Repeated SHS treatments increase the efficiency of targeted mutagenesis in somatic cells of Arabidopsis.(a) Schematic representation of the growth conditions of first‐generation transgenic (T1) plants exposed to heat stress at 37°C. S: stratification at 4°C; d: days.(b) Percentage of GFP‐positive leaf nuclei in different Arabidopsis plants containing the pHTR5::HTR5‐GFP transgene. Ten independent T1 plants for each genotype/growth condition (no CRISPR construct, no CRISPR construct + heat stress, CRISPR construct, and CRISPR construct + heat stress) were assessed. Each bar on the graph represents the nuclei from one individual plant. The numbers 1 to 8 correspond to the plants described in panel (c).(c) Summary of mutation frequencies and recovered alleles identified by sequencing SpCas9‐gRNA HTR5‐1 T1 plants.(d) Percentage of GFP‐positive flower nuclei from different Arabidopsis plants containing the pHTR5::HTR5‐GFP transgene. Five independent plants for each genotype/growth condition were assessed.

Next, we tested if SHS could also increase the efficiency of targeted mutagenesis in the Arabidopsis germline, which is essential for transmitting mutated alleles to the next generation. We used a similar quantitative assay as the one previously described, with the exception that GFP was tagged to MALE‐GAMETE‐SPECIFIC HISTONE H3 (MGH3) (Figure 2(a)), a histone H3 variant highly expressed in the sperm cells of mature tri‐cellular pollen grains of Arabidopsis (Okada et al., 2005; Ingouff et al., 2007). Fluorescence microscopy was used to detect pollen grains with edited sperm cells lacking a functional MGH3‐GFP allele (Figure 2(b)). We tested three sgRNAs targeting different regions of the pMGH3::MGH3‐GFP transgene for these experiments: MGH3‐1 (5′‐end sgRNA), MGH3‐2 (middle sgRNA) and GFP‐3 (3′‐end sgRNA) (Figure 2(a)), and also included a sgRNA targeting BRASSINOSTEROID INSENSITIVE 1 (BRI1) as a negative control. Our results show that T1 plants subjected to repeated SHS produced on average a much higher level (up to 100‐fold difference) of edited pollen grains than T1 plants continuously grown at 22°C (Figure 2(c)). All three sgRNAs targeting the pMGH3::MGH3‐GFP transgene show increased activity by exposure to heat stress at 37°C, with the 5′‐end sgRNA (MGH3‐1) having the largest effect on the efficiency of targeted mutagenesis in the male germline.

Figure 2 Open in figure viewer PowerPoint Heat stress increases the frequency of heritable mutations induced by CRISPR/Cas9.(a) Representation of the pMGH3::MGH3‐GFP reporter gene and the sites targeted by three different sgRNAs.(b) Microscopy image of Arabidopsis pollen grains containing an edited (mutated) or non‐edited pMGH3::MGH3‐GFP transgene.(c) Percentage of GFP‐negative (edited) pollen grains from plants expressing SpCas9 and different sgRNAs. Each bar represents the pollen grains from one individual T1 plant.(d) Percentage of SpCas9‐negative T2 plants with at least one mutated allele in a gene targeted by a matching sgRNA. Numbers 1 and 2 indicate T2 plants represented in the next panel.(e) Percentage of homozygous mutant, biallelic, heterozygous and wild type plants among the T2 plants identified in panel (d). For this experiment, only mutations at the endogenous genes (MGH3 or HTR5) were assessed.

To confirm that mutated alleles were transmitted to the next generation, we collected tissue from T2 plants lacking the pYAO::SpCas9 transgene, extracted genomic DNA and sequenced polymerase chain reaction (PCR) products amplified from targeted loci. We analyzed T2 plants that had lost the pYAO::SpCas9 transgene by random segregation (SpCas9‐negative plants) to ensure that any mutation identified in these plants had been passed down from the male or female germline of the T1 parent plant. For each locus targeted by a specific sgRNA, we analyzed between 10 and 20 T2 plants from selected T1 parents exposed to heat stress. We also performed this analysis on the progeny of T1 plants continuously grown at 22°C, selecting plants with the highest level of GFP‐negative pollen grains (ranging from 2 to 11%). The sgRNAs used in this study (with the exception of the GFP‐3 sgRNA) have two exact matches in the Arabidopsis genome, as both HTR5 and MGH3 have transgenic (GFP tagged) and endogenous sequences in the plant backgrounds that were used. Therefore, we were able to obtain sequencing data from T2 plants for seven different loci (two for the MGH3‐1, MGH3‐2 and HTR5‐1 sgRNAs, and one for the GFP‐3 sgRNA). As shown in Figure 2(d), the percentage of T2 siblings with at least one mutated allele was much higher if its T1 parent had been exposed to repeated SHS, regardless of the sgRNA used or loci targeted by the SpCas9–sgRNA complex. To further characterize the mutation rate increase conferred by repeated SHS treatments, we cloned and sequenced the different PCR products (endogenous loci only) from mutated T2 plants to identify if the plants were heterozygous, biallelic, or homozygous mutants, or were wild type. Most of the T1 plants exposed to SHS produced a large number of T2 plants that were either homozygous or biallelic mutants (Figure 2(e)). These results demonstrate that exposing Arabidopsis to 37°C increases transmission of CRISPR‐induced mutant alleles from the germline, and that a high proportion of biallelic/homozygous T2 mutants can be recovered by exposing T1 parents to SHS.

Multiple studies have shown that CRISPR/Cas9 can generate double‐stranded DNA breaks at off‐target sites (loci where the sgRNA does not match perfectly with the target sequence) in eukaryotic genomes (Wu et al., 2014). In plants, other studies on CRISPR/Cas9 have demonstrated that the frequency of off‐target mutagenesis is either very low or undetectable (Nekrasov et al., 2013; Shan et al., 2013; Upadhyay et al., 2013; Jia and Wang, 2014; Zhang et al., 2014; Zhou et al., 2014), including in Arabidopsis (Li et al., 2013; Feng et al., 2014; Peterson et al., 2016). In light of our previous results showing that targeted mutagenesis is increased in plants exposed to 37°C, we tested if SHS treatments also affect off‐target mutagenesis in Arabidopsis. To do this, we expressed modified pMGH3::MGH3‐GFP transgenes in plants that include one or two substitutions at the site targeted by the MGH3‐1 sgRNA (Figure 3(a)). The substitutions were made at positions 10 and 11 of the target site (20 nt), which fall within the 12‐nt seed region (adjacent to the PAM) important for target specificity of CRISPR/Cas9 (Wu et al., 2014). We assessed the mutation rate of CRISPR/Cas9 on these two modified targets and the unmodified one as a reference, and compared the effect of SHS treatments on mutagenesis at these sites. For this experiment, we analyzed T1 plants by PCR‐amplifying and sequencing the GFP reporter gene using genomic DNA extracted from a rosette leave. This method allowed us to rapidly assess the number of T1 plants (expressing the MGH3‐1 sgRNA and Cas9) with detectable levels of CRISPR/Cas9‐induced mutations in leaf tissue. Our results show that off‐target mutagenesis is not detectable at either of the modified transgenes when Arabidopsis is grown at 22°C (Figure 3(b)), thus confirming previous published reports (Galbraith et al., 1991; Pecinka et al., 2010; Mittler et al., 2012). When plants were subjected to SHS treatments, off‐target mutagenesis was detected on the transgene with one substitution. However, two substitutions at the locus was sufficient to prevent off‐target mutagenesis, indicating that designing sgRNAs that maximize the number of mismatches with potential off‐targets is important when performing CRISPR in plants subjected to conditions that increase the mutation rate. Overall, our results demonstrate that repeated SHS treatments increase both on‐target and off‐target mutagenesis, but undesirable mutations can be minimized by choosing appropriate sgRNAs.

Figure 3 Open in figure viewer PowerPoint Effect of SHS treatments on off‐target mutagenesis by CRISPR/Cas9.(a) Sequence of the region in pMGH3::MGH3‐GFP targeted by the MGH3‐1 sgRNA. One or two substitutions (in red) were introduced in pMGH3::MGH3‐GFP at the site recognized by the MGH3‐1 sgRNA. The PAM motif is underlined.(b) The percentage of T1 plants with detectable mutations in the transgene (wild type (WT), one point mutation or two point mutations). Plants were either grown continuously at 22°C or exposed to four SHS treatments.

One possible mechanism that could explain the increase in CRISPR‐induced mutations when plants are exposed to 37°C is that Cas9 is less active at lower temperatures. The Cas9 homologue most commonly used in CRISPR/Cas9 studies is from S. pyogenes, a bacterium that has an optimal growth temperature of 40°C (Panos and Cohen, 1964). Therefore, the differences in mutation rates may reflect reduced activity of SpCas9 at 22°C as compared with its activity at 37°C which is closer to its natural environment. To test the activity of SpCas9 at different temperatures, we performed in vitro Cas9 cleavage assays using a linear DNA template containing a single 20‐nt target site for the MGH3‐1 sgRNA (Figure 4(a)). We reconstituted SpCas9‐sgRNA MGH3‐1 complexes in vitro, added the DNA template and stopped the reactions after 1 and 5 min for samples incubated at 22°C or 37°C (Figure 4(b)). Our results show that SpCas9 is indeed less active at 22°C than at 37°C, as shown by the 23% decrease in cleaved products after 1 min in reactions incubated at the lower temperature (Figure 4(c)). The amount of cleaved products at 5 min is similar for reactions at 22°C or 37°C, which likely indicates that the maximum reaction yield (approximately 70%) under our conditions has been attained. We also tested if expression levels of SpCas9 and the sgRNA are affected when Arabidopsis plants are exposed to heat stress at 37°C. Our results indicate that levels of SpCas9 are not increased by heat stress, but sgRNA levels are approximately three‐fold higher in plants exposed to 37°C. (Figure 4(d)). As levels of endogenous YAO mRNAs were found not to change in response to heat stress, this strongly suggests that the YAO promoter is not more active at 37°C. If levels of sgRNAs in plants at 22°C are limiting for CRISPR/Cas9 activity, then the in vivo increase of sgRNA upon heat stress could contribute to higher mutation frequencies. Taken together, these findings support our in vivo results showing higher mutagenesis rates by CRISPR/Cas9 in plants exposed to heat stress and suggests that multiple mechanisms are contributing to this effect. These results also suggest that all organisms that can sustain being exposed to 37°C could achieve higher levels of CRISPR‐induced mutagenesis when Cas9 from S. pyogenes is used.

Figure 4 Open in figure viewer PowerPoint SpCas9 is more active at higher temperatures in vitro.(a) Linear DNA template used in the in vitro cleavage assay. Arrows indicate the forward and reverse primers used to amplify the linear DNA template. The MGH3‐1 sgRNA (in red) has one target site on the DNA template.(b) In vitro assay using reconstituted SpCas9–sgRNA MGH3‐1 complexes and the linear template DNA containing a single cleavage site. Reactions were stopped after 1 min or 5 min and loaded on agarose gels to assess cleavage efficiency.(c) Quantification of cleavage efficiency by SpCas9–sgRNA MGH3‐1 at different temperatures. The average and standard error of the mean for five independent experiments are shown. *P = 0.0035.(d) Relative quantification by real‐time PCR of YAO (YAO promoter), SpCas9 (YAO promoter), or MGH3‐1 sgRNA (AtU6‐26 promoter) in plants exposed to heat stress at 37°C for 30 h. The average and standard error of the mean for three biological replicates are shown. *P = 0.0029.

To demonstrate that the effect of temperature on targeted mutagenesis by CRISPR/Cas9 is not specific to Arabidopsis, we regenerated genetically identical Citrus plants expressing the pYAO::SpCas9 transgene and a sgRNA targeting the Citrus phytoene desaturase (CsPDS) gene. Loss‐of‐function mutations or silencing the PDS gene inhibits the production of carotenoids, which results in a white‐colored (albino) phenotype in many plant species, including different Citrus species (Aguero et al., 2014). Regenerated Citrus plants were divided into two groups: plants continuously grown at 24°C and plants exposed to several heat stress treatments (24 h at 37°C, 24 h at 24°C; repeated seven times). Most Citrus plants subjected to heat stress showed a striking phenotype, as all new (i.e. young) aerial tissues produced after exposure to 37°C were almost completely white (albino phenotype) while older tissues remained mostly green (mosaic phenotype) (Figure 5(a)). The phenotypic difference between young and older leaf tissues likely reflects the activity of the YAO promoter, which predominantly drives gene expression in plant tissues with active cell division (Li et al., 2010). As the rate of cell division is much lower in more mature plant tissues, SpCas9 expression is likely too low to induce mutations at the CsPDS gene. In contrast to plants subjected to heat stress, all Citrus plants continuously grown at 24°C did not display any enhancement of the albino phenotype in young tissues (Figure 5(a)). Molecular analysis of the CsPDS alleles in albino tissues recovered after the heat stress treatments showed that all alleles had CRISPR/Cas9‐induced mutations (Figure 5(b)). In contrast, approximately half of the CsPDS alleles were wild type in mosaic leaf tissues (young and old) of plants continuously grown at 24°C, and in older leaf tissue of plants exposed to heat stress. Taken together, our results indicate that exposure to 37°C increases targeted mutagenesis by CRISPR/Cas9 in both Arabidopsis and Citrus.