Qiwei Shan

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).

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.

An efficient TALEN mutagenesis system in rice

Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in rice. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific FokI cleavage domain, are one of the most efficient genome engineering methods developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this paper, we describe a detailed protocol for constructing TALEN expression vectors, assessing nuclease activities in vivo using rice protoplast-based assays, generating and introducing TALEN DNAs into embryogenic calluses of rice and identifying TALEN-generated mutations at targeted genomic sites. Using these methods, T0 rice plants resulting from TALEN mutagenesis can be produced within 4-5 months.

Editing plant genes one base at a time

A new class of gene-editing reagents precisely alters plant genomes without creating a DNA double strand break.

ZFN, TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis: a disconnect between somatic and germinal cells

Breakthroughs in the generation of programmable sequencespecific nucleases (SSNs), such as zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and the RNA-directed nuclease CRISPR-associated protein 9 (Cas9), have greatly increased the ease of plant genome engineering (Voytas, 2013; Malzahn et al., 2017). Programmable SSNs introduce a DNA double-strand break (DSB) at a target site in the genome that must be repaired by one of several endogenous DNA repair pathways. Non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) may result in small insertions and deletions, while homologous recombination (HR), a template-driven repair, is capable of copying information from a homologous donor molecule codelivered with the nuclease. This precise HR-based modification or gene targeting (GT) remains challenging in most plants (Malzahn et al., 2017). Factors limiting plant GT frequencies include the low frequency that HR is used for DNA repair and the difficulty in delivering abundant donor templates to plant cells. GT in Arabidopsis thaliana has proven particularly difficult to optimize; several recent publications have detailed efforts to enhance GT efficiencies but were unable to improve upon existing methods (Shaked et al., 2005; de Pater et al., 2013; Schiml et al., 2014). Here we present data detailing a disconnect in Arabidopsis GT efficiencies between somatic and germinal tissues, our attempts to close that gap, and pitfalls of relying on somatic GT reporters. We first tested ZFN-mediated GT of a RLK gene (At1g53430). We constructed a T-DNA plasmid containing an estrogen inducible ZFN pair targeting RLK, followed by a donor molecule that would introduce an in-frame BAR gene (Fig. S1A). Notably, the ZFN also targets a homologous gene (At1g53440) 10 kb downstream of At1g53430. We predicted that in stably transformed Arabidopsis plants, inducible expression of the ZFN should facilitate HR, resulting in expression of BAR from the promoter of At1g53430 (Fig. S1B). T1 transgenic plants were screened on MS medium supplied with hygromycin (for the transgene) and b-estradiol (to induce ZFN expression) (Fig. S1C). We detected ZFN-induced chromosomal deletions by PCR, confirming that ZFN expression was effectively induced by b-estradiol (Fig. S1D). After transferring transgenic plants from plates to soil, ZFN expression was continuously induced by spraying b-estradiol (10 mM) every two days for approximately one month. Then, T2 seeds from 50 independent T1 parental lines were collected and screened on MS medium containing BASTA, and two T1 lines (#22 and #32) yielded BASTAresistant plants. HR events were detected by PCR in these plants, consistent with the BASTA-resistant phenotype (Fig. S1E). After

CRISPR/Cas: a novel way of RNA-guided genome editing: CRISPR/Cas: a novel way of RNA-guided genome editing

Bacteria and archaea have evolved an adaptive immune system, known as type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which uses short RNA to direct the degradation of target sequences present in invading viral and plasmid DNAs. Recent advances in CRISPR/Cas system provide an improved method for genome editing, showing robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci. It is the latest technology to modify genome DNA specifically and effectively following zinc finger nucleases (ZFNs) and TALE nucleases (TALENs). Compared with ZFNs and TALENs, CRISPR/Cas is much simpler and easier to engineer. This review summarizes recent progress, and discusses the prospects of CRISPR/Cas system, with an emphasis on its structure, principle, applications and potential challenges.CRISPR／Cas系统广泛存在于细菌及古生菌中，是机体长期进化形成的RNA指导的降解入侵病毒或噬菌体DNA的适应性免疫系统。对II型CRISPR／Cas系统的改造使其成为继锌指核酸酶（ZFNs）和TALE核酸酶（TALENs）以来的另一种对基因组进行高效定点修饰的新技术，与ZFNs和TALENs相比，CRISPR／Cas系统更简单，并且更容易操作。文章重点介绍了II型CRISPR／Cas系统的基本结构、作用原理及这一技术在基因组定点修饰中的应用，剖析了该技术可能存在的问题，展望了CRISPR／Cas系统的应用前景，为开展这一领域的研究工作提供参考。