Jinxing Liu

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.

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.

Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.

Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants1, but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.

Biolistic Genetic Transformation of a Wide Range of Chinese Elite Wheat (Triticum aestivum L.) Varieties

Wheat(Triticum aestivum L.)is a major staple food crop worldwide.It is economically important because it can be grown in a wide range of climates and geographic regions,and it has made an enormous contribution to the increase in global food production over the past four decades(Dixon et al.,2009).Wheat is produced on more than 18%of the arable land in the world,and is the most cultivated crop after maize

Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins

This protocol is an extension to: Nat. Protoc. 9, 2395–2410 (2014); doi:10.1038/nprot.2014.157; published online 18 September 2014In recent years, CRISPR/Cas9 has emerged as a powerful tool for improving crop traits. Conventional plant genome editing mainly relies on plasmid-carrying cassettes delivered by Agrobacterium or particle bombardment. Here, we describe DNA-free editing of bread wheat by delivering in vitro transcripts (IVTs) or ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 by particle bombardment. This protocol serves as an extension of our previously published protocol on genome editing in bread wheat using CRISPR/Cas9 plasmids delivered by particle bombardment. The methods we describe not only eliminate random integration of CRISPR/Cas9 into genomic DNA, but also reduce off-target effects. In this protocol extension article, we present detailed protocols for preparation of IVTs and RNPs; validation by PCR/restriction enzyme (RE) and next-generation sequencing; delivery by biolistics; and recovery of mutants and identification of mutants by pooling methods and Sanger sequencing. To use these protocols, researchers should have basic skills and experience in molecular biology and biolistic transformation. By using these protocols, plants edited without the use of any foreign DNA can be generated and identified within 9–11 weeks.

Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits

GW2 is emerging as a key genetic determinant of grain weight in cereal crops; it has three homoeologs (TaGW2-A1, -B1 and -D1) in hexaploid common wheat (Triticum aestivum L.). Here, by analyzing the gene editing mutants that lack one (B1 or D1), two (B1 and D1) or all three (A1, B1 and D1) homoeologs of TaGW2, several insights are gained into the functions of TaGW2-B1 and -D1 in common wheat grain traits. First, both TaGW2-B1 and -D1 affect thousand-grain weight (TGW) by influencing grain width and length, but the effect conferred by TaGW2-B1 is stronger than that of TaGW2-D1. Second, there exists functional interaction between TaGW2 homoeologs because the TGW increase shown by a double mutant (lacking B1 and D1) was substantially larger than that of their single mutants. Third, both TaGW2-B1 and -D1 modulate cell number and length in the outer pericarp of developing grains, with TaGW2-B1 being more potent. Finally, TaGW2 homoeologs also affect grain protein content as this parameter was generally increased in the mutants, especially in the lines lacking two or three homoeologs. Consistent with this finding, two wheat end-use quality-related parameters, flour protein content and gluten strength, were considerably elevated in the mutants. Collectively, our data shed light on functional difference between and additive interaction of TaGW2 homoeologs in the genetic control of grain weight and protein content traits in common wheat, which may accelerate further research on this important gene and its application in wheat improvement.

Genome editing of upstream open reading frames enables translational control in plants

Translational regulation by upstream open reading frames (uORFs) is becoming established as a general mechanism for controlling the amount of protein that is synthesized from downstream primary ORFs (pORFs). We found that genome editing of endogenous uORFs in plants enabled the modulation of translation of mRNAs from four pORFs that are involved in either development or antioxidant biosynthesis. A single-guide RNA that targeted the region harboring a uORF initiation codon can produce multiple mutations. Following uORF editing, we observed varying amounts of mRNA translation in four pORFs. Notably, editing the uORF of LsGGP2, which encodes a key enzyme in vitamin C biosynthesis in lettuce, not only increased oxidation stress tolerance, but also increased ascorbate content by ∼150%. These data indicate that editing plant uORFs provides a generalizable, efficient method for manipulating translation of mRNA that could be applied to dissect biological mechanisms and improve crops.