BackgroundCRISPR-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.ResultsWe 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.ConclusionOur 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.Electronic supplementary materialThe online version of this article (10.1186/s12915-019-0629-5) contains supplementary material, which is available to authorized users.

Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBEC1 and PmCDA1 cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDA1, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

Summary CRISPR ‐Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species‐specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first‐generation single transcript unit ( STU )‐Cas9 system, STU ‐Cas9‐ RZ , which is based on hammerhead ribozyme for processing single guide RNA s (sg RNA s). In this study, we developed two new STU ‐Cas9 systems and one STU ‐Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU ‐Cas9 2.0 systems process the sg RNA s with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU ‐Cas9‐Csy4 and STU ‐Cas9‐ tRNA systems showed more robust genome editing efficiencies than our first‐generation STU ‐Cas9‐ RZ system and the conventional mixed dual promoter system. We further applied the STU ‐Cas9‐ tRNA system to compare two C to T base editing systems based on rAPOBEC 1 and Pm CDA 1 cytidine deaminases. The results suggest STU ‐based Pm CDA 1 base editor system is highly efficient in rice. The STU ‐Cas12a system, based on Cas12a’ self‐processing of a CRISPR RNA (cr RNA ) array, was also developed and demonstrated for expression of a single cr RNA and four cr RNA s. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.

Poplar (Populus) is one of the most important woody plants worldwide. Drought, a primary abiotic stress, seriously affects poplar growth and development. Multiple organellar RNA editing factor (MORF) genes—pivotal factors in the RNA editosome in Arabidopsis thaliana—are indispensable for the regulation of various physiological processes, including organelle C-to-U RNA editing and plasmid development, as well as in the response to stresses. Although the poplar genome sequence has been released, little is known about MORF genes in poplar, especially those involved in the response to drought stress at the genome-wide level. In this study, we identified nine MORF genes in the Populus genome. Based on the structural features of MORF proteins and the topology of the phylogenetic tree, the P. trichocarpa (Ptr) MORF family members were classified into six groups (Groups I–VI). A microsynteny analysis indicated that two (22.2%) PtrMORF genes were tandemly duplicated and seven genes (77.8%) were segmentally duplicated. Based on the dN/dS ratios, purifying selection likely played a major role in the evolution of this family and contributed to functional divergence among PtrMORF genes. Moreover, analysis of qRT-PCR data revealed that PtrMORFs exhibited tissue- and treatment-specific expression patterns. PtrMORF genes in all group were involved in the stress response. These results provide a solid foundation for further analyses of the functions and molecular evolution of MORF genes in poplar, and, in particular, for improving the drought resistance of poplar by genetics manipulation.

FeO nanoparticles synthesized via thermal decomposition in the organic phase have attracted tremendous research interest because of their unique morphology, size dispersion, and crystallinity. However, their poor water dispersibility strongly limited their development in biomedical applications. Therefore, a phase-transfer strategy through which hydrophobic nanoparticles with good performance in the aqueous phase can be obtained is an extremely critical issue. Herein, we present a large-scale, facile, highly efficient strategy for the phase transfer of oleic acid-coated FeO nanoparticles via a reverse-micelle-based oxidative reaction. The reverse micelle system improves the efficiency of the interface oxidative reaction and prevents the aggregation of nanoparticles during the reaction, facilitating the transfer of FeO nanoparticles from the organic phase to the aqueous phase. The transferred FeO nanoparticles are used as a T contrast agent to perform magnetic resonance imaging of CNE2 cells (nasopharyngeal carcinoma cell line). In addition, the free carboxyl groups on the surface of transferred nanoparticles can also be programmed to permit the conjugation of other molecules, in turn allowing nanoparticles to be extended in biological targeting or biological recognition applications. Therefore, this strategy offers a promising platform for the large-scale, highly efficient phase transfer of oleic acid-capped nanoparticles and may become a new paradigm to promote the development of diverse nanoparticles for widespread biomedical applications.