Yanpeng Wang

Targeted genome modification of crop plants using a CRISPR-Cas system

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Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew

Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator–like effector nuclease (TALEN) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.

Genome editing in rice and wheat using the CRISPR/Cas system.

Targeted genome editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are powerful tools for understanding gene function and for developing valuable new traits in plants. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system has recently emerged as an alternative nuclease-based method for efficient and versatile genome engineering. In this system, only the 20-nt targeting sequence within the single-guide RNA (sgRNA) needs to be changed to target different genes. The simplicity of the cloning strategy and the few limitations on potential target sites make the CRISPR/Cas system very appealing. Here we describe a stepwise protocol for the selection of target sites, as well as the design, construction, verification and use of sgRNAs for sequence-specific CRISPR/Cas-mediated mutagenesis and gene targeting in rice and wheat. The CRISPR/Cas system provides a straightforward method for rapid gene targeting within 1–2 weeks in protoplasts, and mutated rice plants can be generated within 13–17 weeks.

Rapid and Efficient Gene Modification in Rice and Brachypodium Using TALENs

In the past few years, the use of sequence-specific nucleases for efficient targeted mutagenesis has provided plant biologists with a powerful new approach for understanding gene function and developing new traits. These nucleases create DNA double-strand breaks at chromosomal targeted sites that are primarily repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathways. NHEJ is often imprecise and can introduce mutations at target sites resulting in the loss of gene function. In contrast, HR uses a homologous DNA template for repair and can be employed to create site-specific sequence modifications or targeted insertions (Moynahan and Jasin, 2010).

Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

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.

Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

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.

Precise base editing in rice, wheat and maize with a Cas9- cytidine deaminase fusion

Targeted base editing in plants without the need for a foreign DNA donor or double-stranded DNA cleavage would accelerate genome modification and breeding in a wide array of crops. We used a CRISPR–Cas9 nickase-cytidine deaminase fusion to achieve targeted conversion of cytosine to thymine from position 3 to 9 within the protospacer in both protoplasts and regenerated rice, wheat and maize plants at frequencies of up to 43.48%.

Establishing a CRISPR–Cas-like immune system conferring DNA virus resistance in plants

CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) is an adaptive immune system in many archaea and bacteria that cleaves foreign DNA on the basis of sequence complementarity. Here, using the geminivirus, beet severe curly top virus (BSCTV), transient assays performed in Nicotiana benthamiana demonstrate that the sgRNA–Cas9 constructs inhibit virus accumulation and introduce mutations at the target sequences. Further, transgenic Arabidopsis and N. benthamiana plants overexpressing sgRNA–Cas9 are highly resistant to virus infection.

High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The 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.

Targeted Mutagenesis in Hexaploid Bread Wheat Using the TALEN and CRISPR/Cas Systems

The use of sequence-specific transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas9) have provided powerful reverse genetic approaches to the targeted modification of genomes in numerous organisms. Both systems have been employed to generate loss-of-function alleles in bread wheat, by targeting multiple and single copies of genes. Here we present protocols for modifying the wheat genome using the two systems. The protocols include the design of TALEN and CRISPR/Cas9 target sites and their construction, evaluation of their activities in protoplasts, transformation of plants, and mutation screening.