Programmed Self-Elimination of the CRISPR/Cas9 Construct Greatly Accelerates the Isolation of Edited and Transgene-Free Rice Plants.

CRISPR gene-editing technology has successfully generated targeted mutations in rice and many other plant species (Ma et al., 2015Ma X. Zhang Q. Zhu Q. Liu W. Chen Y. Qiu R. Wang B. Yang Z. Li H. Lin Y. et al.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.Mol. Plant. 2015; 8: 1274-1284Abstract Full Text Full Text PDF PubMed Scopus (1159) Google Scholar). Assessment of heritability and phenotypic stability of CRISPR-edited plants requires the elimination of the CRISPR construct. The presence of the CRISPR construct makes it difficult to distinguish the mutations transmitted from the previous generation from newly generated mutations by the CRISPR construct at the current generation. The existence of the CRISPR construct also greatly increases the risk of off-target effects, making phenotypic stability a concern. Moreover, removal of the transgenes is likely a prerequisite for gaining regulatory approval of CRISPR-edited crops for commercial applications. Development of an efficient and easy-to-perform technology for the removal of the CRISPR construct after DNA modifications have been completed will have a major impact on using gene-editing technology for crop improvement. Transgene-free, CRISPR-edited plants can be identified by traditional methods such as genetic segregation, backcross, and genotyping, but such methods are very laborious and time consuming. We previously used a mCherry fluorescence marker, which is specifically expressed in Arabidopsis seeds, as a proxy for the presence of CRISPR/Cas9 construct (Gao et al., 2016Gao X. Chen J. Dai X. Zhang D. Zhao Y. An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing.Plant Physiol. 2016; 171: 1794-1800Crossref PubMed Scopus (153) Google Scholar). Such a strategy greatly accelerated our isolation of heritable and Cas9-free Arabidopsis mutants. Furthermore, the focused analysis of Cas9-free T2 Arabidopsis plants revealed that most mutations generated in T1 generation in Arabidopsis were not transmitted to the next generation and that the apparent segregation ratio did not follow the expected Mendelian segregation ratio (Gao et al., 2016Gao X. Chen J. Dai X. Zhang D. Zhao Y. An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing.Plant Physiol. 2016; 171: 1794-1800Crossref PubMed Scopus (153) Google Scholar). Although the fluorescence marker-assisted selection of Cas9-free Arabidopsis plants reduced at least 75% of the workload for identifying transgene-free CRISPR-edited Arabidopsis plants, the strategy was still labor intensive (Gao et al., 2016Gao X. Chen J. Dai X. Zhang D. Zhao Y. An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing.Plant Physiol. 2016; 171: 1794-1800Crossref PubMed Scopus (153) Google Scholar). The fluorescence marker facilitates the identification of the transgene-free plants, but it does not enrich or increase the proportion of transgene-free plants in the T2 generation. Another reported strategy for isolating transgene-free edited plants was to couple the CRISPR construct with an RNA interference element, which targets a herbicide-resistance P450 enzyme. Such a strategy enables herbicide-dependent isolation of transgene-free plants (Lu et al., 2017Lu H.P. Liu S.M. Xu S.L. Chen W.Y. Zhou X. Tan Y.Y. Huang J.Z. Shu Q.Y. CRISPR-S: an active interference element for a rapid and inexpensive selection of genome-edited, transgene-free rice plants.Plant Biotechnol. J. 2017; 15: 1371-1373Crossref PubMed Scopus (54) Google Scholar). However, this strategy still does not enrich the transgene-free plants and requires planting the population. Using Cas9 protein complexed with guide RNAs (RNP) can also generate transgene-free edited plants, but RNP is technically challenging and very few labs have had success with it so far. Moreover, RNP methods cannot use antibiotics/herbicides to provide selection pressure. Consequently many un-mutated plants will also be generated, making it very laborious and time-consuming to identify edited plants. Here, we report the development of a technology that can actively and automatically eliminate any plants containing the CRISPR/Cas9 construct but still allows enough time for the CRISPR/Cas9 construct to perform targeted gene modification before its removal. We employ a pair of suicide transgenes that effectively kills all of the CRISPR/Cas9-containing pollen and embryos produced by T0 plants. Our strategy effectively eliminates the CRISPR/Cas9 transgenes in all of the T1 plants, greatly reducing the labor and time needed to identify transgene-free plants that contain the targeted mutations generated by CRISPR/Cas9. We placed the bacterial BARNASE gene (Mariani et al., 1990Mariani C. Beuckeleer M.D. Truettner J. Leemans J. Goldberg R.B. Induction of male sterility in plants by a chimaeric ribonuclease gene.Nature. 1990; 347: 737Crossref Scopus (624) Google Scholar) under the control of the rice REG2 promoter (Sun et al., 1996Sun J.L. Nakagawa H. Karita S. Ohmiya K. Hattori T. Rice embryo globulins: amino-terminal amino acid sequences, cDNA cloning and expression.Plant Cell Physiol. 1996; 37: 612-620Crossref PubMed Scopus (19) Google Scholar), which has been shown to be expressed during early embryo development (Figure 1A). Expression of the BARNASE gene, which encodes a toxic protein with nuclease activity, is known to kill plant cells. We hypothesized that a REG2-BARNASE cassette would kill any embryos that contain the transgenes. We took advantage of a rice male gametophyte specific lethal protein CMS2, and designed an expression cassette to kill transgene-containing male gametophytes. Expression of the CMS2 gene, which is also called ORFH79, disrupts mitochondria functions during male gametophyte development and causes male sterility (Wang et al., 2006Wang Z. Zou Y. Li X. Zhang Q. Chen L. Wu H. Su D. Chen Y. Guo J. Luo D. et al.Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing.Plant Cell. 2006; 18: 676-687Crossref PubMed Scopus (38) Google Scholar, Hu et al., 2012Hu J. Wang K. Huang W. Liu G. Gao Y. Wang J. Huang Q. Ji Y. Qin X. Wan L. et al.The rice pentatricopeptide repeat protein RF5 restores fertility in hong-lian cytoplasmic male-sterile lines via a complex with the glycine-rich protein GRP162.Plant Cell. 2012; 24: 109-122Crossref PubMed Scopus (212) Google Scholar). We hypothesized that CaMV 35S promoter-driven CMS2 expression would essentially ensure that any CMS2-containing male gametophytes would be destroyed (Figure 1A). We introduced both REG2-BARNASE and 35S-CMS2 expression cassettes into our regular CRISPR/Cas9 plasmid pCXUN-Cas9 (He et al., 2017He Y. Zhang T. Yang N. Xu M. Yan L. Wang L. Wang R. Zhao Y. Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing.J. Genet. Genomics. 2017; 44: 469-472Crossref PubMed Scopus (62) Google Scholar) (Figure 1A). We call the technology that uses suicide transgenes to eliminate CRISPR constructs in plants TKC (Transgene Killer CRISPR). The map of the TKC plasmid is shown in Supplemental Figure 1. We hypothesized that the introduction of our TKC plasmid into rice calli through Agrobacterium-mediated transformation would allow the rice genome to be edited at target sites because the BARNASE toxic protein is not produced in calli or during vegetative growth (Figure 1B). Although the CaMV 35S promoter is a constitutive and strong viral promoter, the CMS2 protein does not kill somatic cells (Zou, 2006Zou Y. Functional Study of the BT Cytoplasmic Male Sterility Gene and Recovery Gene in Rice. South China Agricultural University, 2006Google Scholar). Therefore, we envisioned that our TKC plasmid would allow Cas9 to edit target genes in calli and in vegetative cells of T0 plants (Figure 1B). When T0 plants undergo reproductive growth, the embedded CMS2 and BARNASE expression cassettes will produce toxic proteins that kill male gametophytes and embryos that contain the CRISPR/Cas9 construct, respectively. Therefore, we hypothesized that any seeds obtained from T0 plants transformed with a TKC construct would be transgene free and that some seeds would contain the desired mutations in our target gene (Figure 1B). To test the effectiveness of our strategy, we targeted the rice LAZY1 gene (Li et al., 2007Li P. Wang Y. Qian Q. Fu Z. Wang M. Zeng D. Li B. Wang X. Li J. LAZY1 controls rice shoot gravitropism through regulating polar auxin transport.Cell Res. 2007; 17: 402-410Crossref PubMed Scopus (234) Google Scholar) (Figure 1C), which is known to play an important role in gravitropic response. Loss-of-function lazy1 mutants show a large tiller angle (Figure 1C). The visible phenotypes of lazy1 mutants allowed us to qualitatively evaluate the editing efficiency of our constructs. Among the 65 T0 plants we obtained, 29 plants had obvious tiller angle phenotypes (Figure 1D), indicating that our CRISPR construct was able to generate loss-of-function mutations at the target gene LAZY1. We harvested seeds from each individual T0 plants and analyzed the progeny from 10 independent T0 plants that had a visible lazy1 phenotype for the presence of the transgenes. We used three sets of primers that amplify the Cas9 gene, the CMS2 gene, and the BARNASE gene, respectively (Supplemental Materials and Methods) to detect whether the transgenes were present in the T1 plants. As shown in Supplemental Table 1, all of the T1 plants (total 127, from 10 independent T0 plants with lazy phenotypes and two independent T0 plants without the lazy phenotypes) did not contain the CRISPR construct, demonstrating that our TKC strategy was very effective in eliminating the transgenes. For comparison, when a regular CRISPR/Cas9 construct was used, at least 75% T1 plants harbored the CRISPR/Cas9 construct (Supplemental Table 1). Our initial detection of potential mutations in the T1 plants was based on PCR and restriction digestion (Figure 1C and 1D). The target sequence contains a PstI site near the PAM sequence (Figure 1C). Small deletion/insertion at the target sequence would likely eliminate the restriction site, rendering PstI resistance (PstIr). We noticed that if the first “C” at the PstI site was deleted, the PstI site would not be destroyed (Figure 1C). As an example, we analyzed the 20 T1 plants that were offspring of the T0 plant #40 (Figure 1D). Interestingly, the segregation ratio of PstIr plants and PstI-sensitive plants (PstIs) was 6:8:6 (PstIr/PstIr/s/PstIs), which deviated significantly from the expected Mendelian ratio. We sequenced all of the 110 T1 plants that were the progeny of 10 independent T0 plants with the lazy1 phenotypes (Supplemental Table 1, Figure 1E, and Supplemental Figure 2). Every single plant contained mutations at the target site and they were either homozygous or bi-allelic. We observed 100% efficacy in eliminating both the LAZY1 functions and the transgenes, demonstrating the power of this technology. We further analyzed the segregation patterns and made some interesting observations. All progeny from T0 plant #34 were homozygous with a deletion of a single “C,” suggesting that the T0 plant was already homozygous. We detected two types mutations in T1 plants from T0 #30: a deletion of a “C” and a deletion of a “T” (Figure 1E). We do not think that the T0 #30 was bi-allelic because we did not observe any bi-allelic T1 progeny. In addition, the “C” deletion was more prominent, suggesting that #30 T0 plant was mosaic. Without eliminating the CRISPR construct, it would be much more difficult to interpret the results from the #30 T0 plant. The mosaic nature of T0 plants was more obvious for the plant #3 (Figure 1E), which produced three different alleles: an insertion of an “A,” a deletion of a “C,” and a deletion of four base pairs. The first two types were predominant, accounting for 95% of the T1 plants. The third type would probably have been missed or interpreted as newly created by Cas9 if we did not eliminate the CRISPR construct. For some apparently bi-allelic T0 plants, we noticed that the segregation ratios deviated significantly from the expected Mendelian segregation ratio (Figure 1E). It is likely that the small number of plants analyzed may obscure the segregation ratio. We also analyzed the progenies from T0 plants that did not show the lazy1 phenotype (Supplemental Figure 3). All of the T1 plants (total of 17 plants from two independent T0 plants) analyzed were also transgene free (Supplemental Table 1). Interestingly, the T1 plants were also edited at the target site (Supplemental Figure 3), but some of the mutations were a deletion of 3 bp or a substitution of a “C” with an “A” (Supplemental Figure 3). Such mutations likely did not disrupt the function of LAZY1 proteins, accounting for the observed non-lazy1 phenotypes. To further test the applicability of our TKC technology, we constructed a TKC-D3 plasmid that targets the DWARF3 (D3) gene, which is involved in strigolactone response. The progenies (total 59 plants) from two independent T0 plants were also transgene-free. Moreover, all of them contained mutations in the target site (Supplemental Figure 4). We have clearly demonstrated that the suicide transgenes, BARNASE and CMS2, can be harnessed to eliminate the CRISPR constructs within a single generation without compromising the gene-editing efficiency of CRISPR/Cas9. Our technology greatly reduced the time and labor needed for isolating transgene-free, CRISPR-edited rice plants. Another advantage of our strategy is that transgene-containing pollen or seeds are not released to the environment. Our strategy can be easily adopted for other plant species that can be transformed through tissue culture, although it probably will not work in Arabidopsis because of the floral-dipping transformation. The strategy will be even more beneficial for crops that have long life cycles and produce fewer seeds. This work was supported by a National Transgenic Science and Technology Program (2016ZX08010002) grant to R.W., Huazhong Agricultural University Scientific & Technological Self-Innovation Foundation (2012YB04) to Y.H., and a startup fund from the Huazhong Agricultural University.