Yanfei Mao

The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation

The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9-induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9-induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large-scale off-targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off-target sites of a large number of T0 plants, low-frequency mutations were found in only one off-target site where the sequence had 1-bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.

Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

Significance The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been used to generate targeted gene editing in plants. However, it is not known whether CRISPR/Cas-induced gene modifications in plants occur in somatic cells only or whether some or all of the modifications can enter the germ line to become heritable. Through systematic and multigenerational analysis, this study demonstrates that although the majority of gene modifications detected in the first generation CRISPR/Cas transgenic Arabidopsis plants were somatic mutations only, heritable mutations could be found in subsequent generations. In addition, deep sequencing of CRISPR/Cas-modified Arabidopsis genomes did not detect any off-targets. The work demonstrates that the CRISPR/Cas method can effectively create specific gene modifications in planta that are stably transmitted through the germ line to future generations. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.

Application of the CRISPR-Cas system for efficient genome engineering in plants.

Dear Editor, Recently, engineered endonucleases, such as Zinc-Finger Nucleases (ZFNs) (Carroll, 2011), Transcription Activator-Like Effector Nucleases (TALENs) (Mahfouz et al., 2011; Li et al., 2012), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) systems (Cong et al., 2013) have been successfully used for gene editing in a variety of species. These systems generate double-strand breaks (DSBs) at target loci to drive site-specific DNA sequence modifications. The modifications include sequence insertion and deletion and other mutations in the host genomes via the error-prone non-homologous end joining (NHEJ) pathway or sequence correction or replacement through the error-free homologous recombination (HR) pathway (Symington and Gautier, 2011). Here, we show that the CRISPR–Cas system can be applied to generate targeted gene mutations and gene corrections in plants, and the system can also be readily engineered to achieve deletion of large DNA fragments and for multiplex gene editing in plants. Both ZFNs and TALENs have tandem repeats in their DNA-binding domains that can be engineered to recognize specific DNA sequences; the resulting chimeric nucleases can thus be guided to the desired target sequences in the genome to generate DSBs. For each target site, a new ZFN or TALEN chimeric protein needs to be engineered to recognize the target. This has been a major hurdle in the wide use of these two gene-editing systems because engineering a new protein is no trivial task. In comparison, the newly developed CRISPR–Cas system uses a short single guide RNA (sgRNA) to direct the Cas9 endonuclease to complementary target DNA (Gaj et al., 2013), so only a new sgRNA is needed for a new target site. This system thus greatly simplifies the gene-editing process and widens target-site selection. An additional requirement for the Cas9 nuclease activity is the presence of the protospacer-associated motif (PAM) NGG downstream of the target site. This requirement is an important consideration in target-site selection (Jinek et al., 2012). Several expression vectors were constructed for CRISPR–Cas-based gene editing in Arabidopsis and rice, in which the designed sgRNAs and optimized SpCas9 (Cong et al., 2013) were driven by AtU6 or OsU6 and AtUBQ, OsUBQ, or CaMV 35S promoters, respectively (Supplemental Figure 1). A transient expression system was developed in Arabidopsis protoplasts according to an earlier study (Zhang et al., 2013) to assess the activity of the CRISPR–Cas construct psgR–Cas9–At. The yellow fluorescent protein (YFP)-based reporter contained two partially overlapped YFP fragments that were interrupted by a multiple recognition site (MRS). Three different nucleases, I-SceI, gdTALEN, and CRISPR–Cas, were designed to target the MRS region (Supplemental Figure 2). When the MRS sequence is recognized and cleaved, a functional copy of the YFP gene could be restored through the HR pathway in the cells, so that the efficiency of these endonucleases can be estimated by counting the number of cells emitting yellow fluorescent light using flow cytometry. In an optimized experiment, 11.0% sgR–MRS/YFFP co-transfected protoplasts showed fluorescence—a frequency lower than the 18.8% for gdTALEN but comparable to the 12.5% for I-SceI (Supplemental Figure 2). These results suggested that the CRISPR–Cas system was functional in generating DSBs and triggering gene correction in plant cells. To test the importance of the PAM sequence for target recognition in plants, an improper sgRNA (sgR-MRS*) with a shifted PAM sequence (from GGG to GGA) was used to target the MRS in the YFFP reporter (Supplemental Table 1 and Supplemental Figure 2). The proportion of YFP florescent cells in the sgR-MRS*-targeted protoplasts was 5.4%, compared to the 11% for the sgR-MRS target with a correct PAM. Thus, the altered PAM sequence greatly reduced but did not abolish the activity of CRISPR–Cas9, suggesting that, although PAM is important, it is not absolutely required for the function of CRISPR–Cas in plant cells. The sgRNA and Cas9 expression cassettes were cloned into a binary vector that contains a nonfunctional GUUS reporter (Figure 1A and Supplemental Figure 1) for Agrobacterium-mediated transformation. Among 44 T1 transgenic Arabi dopsis plants tested, five showed a GUS signal in their cotyledons (Figure 1B). We did not see any plant organ with a uniform GUS signal, and observed GUS expression in one guard cell but not in the other one in the same stoma (Figure 1B), indicating that the CRISPR–Cas induced cleavage and HR repair events happened in individual cells. Using a SURVEYOR assay, we found that 35 of the 44 plants, including three of the five GUS-positive lines, had mutations at the target site (Figure 1C and Supplemental Table 2). These data suggest that CRISPR–Cas-generated DSB can be repaired via both NHEJ and HR (Supplemental Figure 3), but NHEJ is the dominant DNA repair pathway in plants. Cloning and sequencing of the PCR products from three of the lines with mutations in the target site revealed that deletions were more abundant and longer in size (6–25bp) than insertions (1–2bp) (Supplemental Figure 4). Figure 1. The CRISPR–Cas9 System Induces Efficient Targeted Gene Editing and Correction in Plants. We then tried to simultaneously target two sites in the Arabidopsis genome using a CRISPR–Cas9 construct that contained two sgRNA expression cassettes. The magnesium-chelatase subunit I (CHLI) genes, CHLI1 (At4g18480) and CHLI2 (At5g45930), were selected for the test. The chli1 chli2 double mutant was albino, while mutations in either gene alone resulted in pale-green plants (Huang and Li, 2009). Two sgRNAs, each targeting one of the CHLI genes, were placed into the p2xsgR–Cas-At vector for Arabidopsis transformation (Figure 1D and Supplemental Table 2). Out of 60 T1 transformants, 23 could not survive to have true leaves because of their severe albino phenotype. The rest grew slowly and most of them exhibited a mosaic leaf color phenotype (Figure 1E). Three transgenic lines with different leaf colors were selected to test for mutations by the SURVEYOR assay and sequencing (Figure 1E). All three lines were found to harbor mutations in both genes (Figure 1G). In the dark-green and pale-green lines, 60% of the CHLI1 PCR products were wild-type, while, in the albino line, the proportion of wild-type CHLI1 was reduced to 14% (Supplemental Figure 5). In contrast, the proportions of wild-type CHLI2 in all three lines were similar, between 10% and 20% (Supplemental Figure 6). The proportion of small insertion and deletion (indels) ( 5bp) were only found in the albino line. The rate of long deletions in CHLI2 increased with the severity of the albino phenotype. In addition, longer deletions were more abundant in CHLI2 than in CHLI1, and more prevailing in the albino line than in the green lines (Figure 1F). Transgenic line #17 had one cotyledon green while the other one had half green and half yellow (Figure 1E). Later, new true leaves emerged as albino, suggesting that double gene mutation events occurred in the shoot apical meristem. The data showed that CRISPR–Cas could be used for multiplex gene editing in plants and suggested that the efficiency of CRISPR–Cas varied at different target sites. We also tried to modify the Arabidopsis TT4 (At5g13930) gene. Two sites separated by 230bp in the TT4 gene were selected for gene editing. Among the 58 T1 transgenic seedlings, 89% of them had mutations at the first site, 84% at the second site, and 74% at both sites (Supplemental Table 2). Furthermore, an amplification fragment length polymorphism (AFLP) assay was performed with primers flanking the two target sites (Figure 1I). Fifteen plants (26%) showed a short PCR product of about 335bp in addition to the 566-bp wild-type product, indicating that DSBs were created at both sites, which resulted in the deletion of the fragment between the two sites (Figure 1H). Sequencing of the PCR products from 11 of the 15 seedlings showed that most of the mutations were small indels (61%) and relatively large deletions (32%), while duplications (2.5%) and inversions (4%) were occasionally found (Supplemental Figures 7 and 8). To test whether the CRISPR–Cas9 system functions in a monocot, we used the system to target the OsMYB1 gene (LOC_Os1g12700) of rice. Twenty T0 transgenic rice plants were recovered (Figure 1K). Out of 20 T0 transgenic rice plants, 10 were found to be wild-type for the OsMYB gene and 10 contained mutations in the target site (Figure 1J). Sequencing revealed that the plants were mosaics of various deletions and small insertions as well as the wild-type allele (Supplemental Figure 9). Our results demonstrated that the CRISPR–Cas system was efficient in targeted genome engineering in both monocot and dicot plants. In general, the system generated detectable mutations at a frequency of 50–89% for a single locus and 68–74% for double loci in plants (Supplemental Table 2). Our results suggest that CRISPR–Cas can be used not only for targeted gene mutagenesis, but also for gene correction and deletion of large genomic fragments.

Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis

The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.

Multiplex Gene Editing in Rice Using the CRISPR-Cpf1 System

The class 2/type II clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been used successfully for simultaneous modification of multiple loci in plants. Two general strategies have been applied to coexpress multiple single guide RNAs (sgRNAs) to achieve multiplex gene editing in plant cells. One is to construct the multiple guide RNA expression cassettes into separate plasmids when direct gene delivery methods are adopted, such as biolistic bombardment and PEG-mediated protoplast transfection (Shan et al., 2013).

TALEN‐mediated targeted mutagenesis produces a large variety of heritable mutations in rice

CRISPR/Cas9 and TALEN are currently the two systems of choice for genome editing. We have studied the efficiency of the TALEN system in rice as well as the nature and inheritability of TALEN-induced mutations and found important features of this technology. The N287C230 TALEN backbone resulted in low mutation rates (0-6.6%), but truncations in its C-terminal domain dramatically increased efficiency to 25%. In most transgenic T0 plants, TALEN produced a single prevalent mutation accompanied by a variety of low-frequency mutations. For each independent T0 plant, the prevalent mutation was present in most tissues within a single tiller as well as in all tillers examined, suggesting that TALEN-induced mutations occurred very early in the development of the shoot apical meristem. Multigenerational analysis showed that TALEN-induced mutations were stably transmitted to the T1 and T2 populations in a normal Mendelian fashion. In our study, the vast majority of TALEN-induced mutations (~81%) affected multiple bases and ~70% of them were deletions. Our results contrast with published reports for the CRISPR/Cas9 system in rice, in which the predominant mutations affected single bases and deletions accounted for only 3.3% of the overall mutations.

Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-Based CRISPR/Cas9 System

Rice (Oryza sativa) is the staple food for more than half of the worlds population. Technologies enabling precise and efficient DNA knock-in or replacement, hereinafter referred to as KI, have the potential to revolutionize the generation of crops by precision molecular breeding. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 (CRISPR/Cas9) has recently emerged as a promising genome editing tool allowing precise genomic manipulation in rice and other crops. However, due to the prevalence of non-homologous end joining (NHEJ) over homology-directed repair (HDR) in the repair of CRISPR/Cas9-induced double-strand breaks (DSBs), this genome editing tool has been mostly used to generate random insertions and deletions (Indels) in precise genomic locations in plants (Cong et al., 2013; Feng et al., 2013; Miao et al., 2013; Shan et al., 2013; Ma et al., 2015; Xie et al., 2015; Gao et al., 2016).

Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits

Significance Plant microRNAs (miRNAs) control intricate gene regulatory networks and have been implicated in important developmental switches and stress responses. Plant miRNAs have recently emerged as promising targets for crop improvement because they can control complex agronomic traits; however, functional studies using reverse genetics have been hampered by practical difficulties. We have silenced 35 miRNA families in rice to generate a resource for discovering new functions of miRNAs and targets of agronomic improvements. As a proof of concept, we show that manipulation of a promising miRNA, miRNA398, leads to important yield improvements. Our findings also reveal important agronomic roles for several miRNAs. Improvements in plant agricultural productivity are urgently needed to reduce the dependency on limited natural resources and produce enough food for a growing world population. Human intervention over thousands of years has improved the yield of important crops; however, it is increasingly difficult to find new targets for genetic improvement. MicroRNAs (miRNAs) are promising targets for crop improvement, but their inactivation is technically challenging and has hampered functional analyses. We have produced a large collection of transgenic short tandem target mimic (STTM) lines silencing 35 miRNA families in rice as a resource for functional studies and crop improvement. Visual assessment of field-grown miRNA-silenced lines uncovered alterations in many valuable agronomic traits, including plant height, tiller number, and grain number, that remained stable for up to five generations. We show that manipulation of miR398 can increase panicle length, grain number, and grain size in rice. In addition, we discovered additional agronomic functions for several known miRNAs, including miR172 and miR156. Our collection of STTM lines thus represents a valuable resource for functional analysis of rice miRNAs, as well as for agronomic improvement that can be readily transferred to other important food crops.

TCP24 modulates secondary cell wall thickening and anther endothecium development.

miR319-targeted TCP genes are believed to regulate cell division in leaves and floral organs. However, it remains unknown whether these genes are involved in cell wall development. Here, we report that TCP24 negatively regulates secondary wall thickening in floral organs and roots. The overexpression of the miR319a-resistant version of TCP24 in Arabidopsis disrupted the thickening of secondary cell walls in the anther endothecium, leading to male sterility because of arrested anther dehiscence and pollen release. Several genes linked to secondary cell wall biogenesis and thickening were down-regulated in these transgenic plants. By contrast, the inhibition of TCP24 using the ectopic expression of a TCP24-SRDX repressor fusion protein, or the silencing of TCP genes by miR319a overexpression, increased cell wall lignification and the enhanced secondary cell wall thickening. Our results suggest that TCP24 acts as an important regulator of secondary cell wall thickening and modulates anther endothecium development.

Efficient Generation of diRNAs Requires Components in the Posttranscriptional Gene Silencing Pathway

It has been reported that double-stranded break (DSB)-induced small RNAs (diRNAs) are generated via the RNA-directed DNA methylation pathway and function in DSB repair in Arabidposis. However, important questions remain regarding the biogenesis and function of diRNAs. Here, we used CRISPR/Cas9- or TALEN-triggered DSBs to characterize diRNAs in Arabidopsis and rice. We found that 21-nt diRNAs were generated from a 35S promoter::GU-US reporter transgene targeted by CRISPR/Cas9. Unexpectedly, Pol II transcription of the transgene was required for efficient diRNA production and the level of diRNA accumulation correlated with the expression level of the transgene. diRNAs were not detected from CRISPR/Cas9- or TALEN-induced DSBs within the examined endogenous genes in Arabidopsis or rice. We also found that DCL4 and RDR6 that are known to be involved in posttranscriptional gene silencing were required to generate diRNAs. Our results suggest that DSBs are necessary but not sufficient for efficient diRNA generation and a high level of diRNAs is not necessary for DSB repair.