An Agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T₀ transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T₁ progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.     

Gene editing of the wheat homologs of TONNEAU1‐recruiting motif encoding gene affects grain shape and weight in wheat

Grain size and weight are important components of a suite of yield‐related traits in crops. Here, we showed that the CRISPR‐Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1‐recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double‐copy mutant showing larger effect than the respective single copy mutants. The TaGW7‐centered gene co‐expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co‐localization of TaGW7 with α‐ and β‐tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7‐associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR‐Cas9 system with cross‐species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.     

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.     

Knockout of two BnaMAX1 homologs by CRISPR/Cas9‐targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.)

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T₀ plants were edited very efficiently (56.30%–67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi‐allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T₁ and T₂ progeny. We also obtained transgene‐free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off‐target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi‐dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi‐dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.     

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops.High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat.The emerging effective genome editing tool,CRISPR/Cas9 system,makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles.To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat,we compared four different RNA polymerase(Pol) Ⅲ promoters(TaU3 p,TaU6 p,OsU3 p,and OsU6 p) and three types of sgRNA scaffold in the protoplast system.We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective.The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles,whose orthologs,OsNP1 in rice and ZmIPE1 in maize,encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility.Triple homozygous mutations in TaNP1 genes result in complete male sterility.We further demonstrated that anyone wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility.Taken together,this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.     

Patterns of CRISPR/Cas9 activity in plants,animals and microbes

The CRISPR/Cas9 system and related RNA‐guided endonucleases can introduce double‐strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on‐target mutations), the targeting accuracy (likelihood of off‐target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species‐dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome‐editing tools.     

CRISPR/Cas9‐mediated gene knockout in the ascidian Ciona intestinalis

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on‐target site. CRISPR/Cas9‐mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.     

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Processing of double‐stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL‐effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi‐allelic double mutant for the two soya bean paralogous Double‐stranded RNA‐binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9‐generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ‐line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer‐like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer‐like3 gene and the GmHen1a gene was observed in the T₀ generation, but these mutations failed to transmit to the T₁ generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole‐genome sequencing to reveal a spectrum of non‐germ‐line‐targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.     

Base editing with high efficiency in allotetraploid oilseed rape by A3A‐PBE system

CRISPR/Cas‐base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A‐PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base‐editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base‐edited plants of ALS conferred high herbicide resistance, while base‐edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T₀ to T₁ generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off‐target sites, large‐scale SNP variations were determined through whole‐genome sequencing between some base‐edited and wild‐type plants. These results revealed that A3A‐PBE base‐editing system could effectively convert C to T substitution with high‐editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.     

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium‐mediated transformation to 5–6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9‐mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9‐mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.     

High‐efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system

The base‐editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum‐Base Editor 3 (GhBE3) base‐editing system has been developed to create single‐base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32‐GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an ‘editing window’, approximately six‐nucleotide windows of ?17 to ?12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off‐target sites predicted by CRISPR‐P and Cas‐OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole‐genome sequencing analyses on two GhCLA‐edited and one wild‐type plants with about 100× depth showed that no bona fide off‐target mutations were detectable from 1500 predicted potential off‐target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.     

One‐step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9‐expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off‐target effects were observed from off‐target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene‐disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild‐type CHO‐S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.     

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots

As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean.     

CRISPR/Cas9 in rice can induce new mutations in later generations,leading to chimerism and unpredicted segregation of the targeted mutation

CRISPR/Cas9 is a novel tool for targeted mutagenesis and is applicable to plants, including rice. Previous reports on CRISPR/Cas9 in rice have demonstrated that target mutations are transmitted to the next generation in accordance with Mendelian law, but heritability of the target mutation and the role of inherited Cas9 gene have not been fully elucidated. Here, we targeted the rice phytoene desaturase gene, mutants of which exhibit an albino phenotype, by using CRISPR/Cas9 and analyzed segregation of target mutations. Agrobacterium-mediated methods using immature embryos successfully transformed a CRISPR/Cas9 system into five rice cultivars and subsequently induced mutation. Unpredicted segregations, with more mutants than theoretically predicted, were frequently found in T₁ plants from monoallelic T₀ mutants. Chimeric plants with both biallelic and monoallelic mutated cells were also observed in the T₁. Next, we followed segregation of a target mutation in the T₂ from monoallelic T₁ mutants. When T₁ mutants possessed Cas9, unpredicted segregations of the target mutation and chimeric plants were observed again in the T₂. When T₁ mutants did not possess Cas9, segregation of the target mutations followed Mendelian law and no chimeric plants appeared in the T₂. T₂ mutants with Cas9 had mutations different from the original ones found in T₀. Our results indicated that inherited Cas9 was still active in later generations and could induce new mutations in the progeny, leading to chimerism and unpredicted segregation. We conclude that Cas9 has to be eliminated by segregation in T₁ to generate homozygous mutants without chimerism or unpredicted segregation.     

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.     

Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9

Microalgae are promising feedstock for biofuels yet mechanistic probing of their cellular network and industrial strain development have been hindered by lack of genome‐editing tools. Nannochloropsis spp. are emerging model microalgae for scalable oil production and carbon sequestration. Here we established a CRISPR/Cas9‐based precise genome‐editing approach for the industrial oleaginous microalga Nannochloropsis oceanica, using nitrate reductase (NR; g7988) as example. A new screening procedure that compares between restriction enzyme‐digested nested PCR (nPCR) products derived from enzyme‐digested and not‐digested genomic DNA of transformant pools was developed to quickly, yet reliably, detect genome‐engineered mutants. Deep sequencing of nPCR products directly amplified from pooled genomic DNA revealed over an 1% proportion of 5‐bp deletion mutants and a lower frequency of 12‐bp deletion mutants, with both types of editing precisely located at the targeted site. The isolated mutants, in which precise deletion of five bases caused a frameshift in NR translation, grow normally under NH₄Cl but fail to grow under NaNO₃, and thus represent a valuable chassis strain for transgenic‐strain development. This demonstration of CRISPR/Cas9‐based genome editing in industrial microalgae opens many doors for microalgae‐based biotechnological applications.