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Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.

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SUMMARYThe ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

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The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.

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