CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, ...but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing.
We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis.
Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.
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Summary
CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region ...that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Recent advances in the genome-editing tools have demonstrated a great potential for accelerating functional genomics and crop trait improvements, but the low efficiency and genotype dependence in ...plant transformation hinder practical applications of such revolutionary tools. Morphogenic transcription factors (MTFs) such as Baby boom, Wuschel2, GROWTH-REGULATING FACTOR5, GROWTH-REGULATING FACTOR4 and its cofactor GRF-INTERACTING FACTOR1, and Wuschel-homeobox 5 related have been shown to greatly enhance plant transformation efficiency and expand the range of amenable species and genotypes. This review will summarize recent advancements in plant transformation technologies with an emphasis on the strategies developed for genotype-flexible transformation methods utilizing MTFs for both monocots and dicot plant species. We highlight several breakthrough studies that demonstrated a wide range of applicability.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred ...lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16-22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7-10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the
ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.
Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a ...methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT-2020. In addition, the same approach is applied to test the efficacy of multiple gRNAs for CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.
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Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with
/G418 selection and a ...compatible helper plasmid can efficiently transform maize inbred B104 using our rapid
-mediated transformation method. In this work, we implemented the non-integrating
(
) T-DNA vector method for
-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating
(NIW) T-DNA vector-assisted transformation method uses two
strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor
co-integration into the maize genome, we combined the maize
expression cassette driven by a strong constitutive promoter with a new visible marker
, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for
gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing.
Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of
identified as teosinte,
ssp.
is the most ...closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the maize domestication process and facilitate breeding for future crop improvement. However, a protocol of genetic transformation, which is essential for gene functional analyses, is not available in teosinte. In this study, we report the establishment of a robust callus induction and regeneration protocol using whorl segments of seedlings germinated from mature seeds of
. We also report, for the first time, the production of fertile, transgenic teosinte plants using the particle bombardment. Using herbicide resistance genes such as mutant acetolactate synthase (
) or bialaphos resistance (
) as selectable markers, we achieved an average transformation frequency of 4.17% (percentage of independent transgenic events in total bombarded explants that produced callus). Expression of visual marker genes of red fluorescent protein
and β-glucuronidase (
) could be detected in bombarded callus culture and in T1 and T2 progeny plants. The protocol established in this work provides a major enabling technology for research toward the understanding of this important plant in crop domestication.
Agrobacterium tumefaciens is a plant pathogen that has the natural ability of delivering and integrating a piece of its own DNA into plant genome. Although bacterial non-coding RNAs (ncRNAs) have ...been shown to regulate various biological processes including virulence, we have limited knowledge of how Agrobacterium ncRNAs regulate this unique inter-Kingdom gene transfer. Using whole transcriptome sequencing and an ncRNA search algorithm developed for this work, we identified 475 highly expressed candidate ncRNAs from A. tumefaciens C58, including 101 trans-encoded small RNAs (sRNAs), 354 antisense RNAs (asRNAs), 20 5' untranslated region (UTR) leaders including a RNA thermosensor and 6 riboswitches. Moreover, transcription start site (TSS) mapping analysis revealed that about 51% of the mapped mRNAs have 5' UTRs longer than 60 nt, suggesting that numerous cis-acting regulatory elements might be encoded in the A. tumefaciens genome. Eighteen asRNAs were found on the complementary strands of virA, virB, virC, virD, and virE operons. Fifteen ncRNAs were induced and 7 were suppressed by the Agrobacterium virulence (vir) gene inducer acetosyringone (AS), a phenolic compound secreted by the plants. Interestingly, fourteen of the AS-induced ncRNAs have putative vir box sequences in the upstream regions. We experimentally validated expression of 36 ncRNAs using Northern blot and Rapid Amplification of cDNA Ends analyses. We show functional relevance of two 5' UTR elements: a RNA thermonsensor (C1_109596F) that may regulate translation of the major cold shock protein cspA, and a thi-box riboswitch (C1_2541934R) that may transcriptionally regulate a thiamine biosynthesis operon, thiCOGG. Further studies on ncRNAs functions in this bacterium may provide insights and strategies that can be used to better manage pathogenic bacteria for plants and to improve Agrobacterum-mediated plant transformation.
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The metabolome and transcriptome of the maize-infecting fungi Ustilago maydis and Fusarium verticillioides were analyzed as the two fungi interact. Both fungi were grown for 7 days in liquid medium ...alone or together in order to study how this interaction changes their metabolomic and transcriptomic profiles. When grown together, decreased biomass accumulation occurs for both fungi after an initial acceleration of growth compared to the biomass changes that occur when grown alone. The biomass of U. maydis declined most severely over time and may be attributed to the action of F. verticillioides, which secretes toxic secondary metabolites and expresses genes encoding adhesive and cell wall-degrading proteins at higher levels than when grown alone. U. maydis responds to cocultivation by expressing siderophore biosynthetic genes and more highly expresses genes potentially involved in toxin biosynthesis. Also, higher expression was noted for clustered genes encoding secreted proteins that are unique to U. maydis and that may play a role during colonization of maize. Conversely, decreased gene expression was seen for U. maydis genes encoding the synthesis of ustilagic acid, mannosylerythritol D, and another uncharacterized metabolite. Ultimately, U. maydis is unable to react efficiently to the toxic response of F. verticillioides and proportionally loses more biomass. This in vitro study clarifies potential mechanisms of antagonism between these two fungi that also may occur in the soil or in maize, niches for both fungi where they likely interact in nature.
Summary
Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter‐kingdom gene transfer capability made ...Agrobacterium an essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such as Agrobacterium‐host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA‐guided integrase system for Agrobacterium genome engineering. In this work, we demonstrate that INsertion of Transposable Elements by Guide RNA–Assisted TargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre‐loxP recombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies for Agrobacterium species and their gene functional analyses.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
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