State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state‐of‐the‐art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.     

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.     

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.     

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‐mediated knockout of Ms1 enables the rapid generation of male‐sterile hexaploid wheat lines for use in hybrid seed production

The development and adoption of hybrid seed technology have led to dramatic increases in agricultural productivity. However, it has been a challenge to develop a commercially viable platform for the production of hybrid wheat (Triticum aestivum) seed due to wheat's strong inbreeding habit. Recently, a novel platform for commercial hybrid seed production was described. This hybridization platform utilizes nuclear male sterility to force outcrossing and has been applied to maize and rice. With the recent molecular identification of the wheat male fertility gene Ms1, it is now possible to extend the use of this novel hybridization platform to wheat. In this report, we used the CRISPR/Cas9 system to generate heritable, targeted mutations in Ms1. The introduction of biallelic frameshift mutations into Ms1 resulted in complete male sterility in wheat cultivars Fielder and Gladius, and several of the selected male‐sterile lines were potentially non‐transgenic. Our study demonstrates the utility of the CRISPR/Cas9 system for the rapid generation of male sterility in commercial wheat cultivars. This represents an important step towards capturing heterosis to improve wheat yields, through the production and use of hybrid seed on an industrial scale.     

Lignin characterization of rice CONIFERALDEHYDE 5‐HYDROXYLASE loss‐of‐function mutants generated with the CRISPR/Cas9 system

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5‐hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1‐knockdown rice lines, which were produced via an RNA‐interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss‐of‐function mutants of OsCAld5H1 using the CRISPR/Cas9‐mediated genome editing system. Homozygous OsCAld5H1‐knockout lines harboring anticipated frame‐shift mutations in OsCAld5H1 were successfully obtained. A series of wet‐chemical and two‐dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ‐p‐coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non‐γ‐p‐coumaroylated units, whereas grass‐specific γ‐p‐coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non‐γ‐p‐coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass‐specific γ‐p‐coumaroylated S units in rice.     

Creation of zebrafish knock‐in reporter lines in the nefma gene by Cas9‐mediated homologous recombination

CRISPR/Cas9‐based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)‐induced mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock‐ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9‐targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence. The resulting knock‐in embryos express the added gene (fluorescent reporter or KalTA4 transactivator) specifically in the populations of neurons that express nefma, making them convenient tools for research on these populations.     

CRISPR/Cas9‐mediated genome modification in the mollusc,Crepidula fornicata

The discovery and application of the CRISPR/Cas9 genome editing method has greatly enhanced the ease with which transgenic manipulation can occur. We applied this technology to the mollusc, Crepidula fornicata, and have successfully created transgenic embryos expressing mCherry fused to endogenous β‐catenin. Specific integration of the fluorescent reporter was achieved by homologous recombination with a β‐catenin‐specific donor DNA containing the mCherry coding sequence. This fluorescent gene knock‐in strategy permits in vivo observations of β‐catenin expression during embryonic development and represents the first demonstration of CRISPR/Cas9‐mediated transgenesis in the Lophotrochozoa superphylum. The CRISPR/Cas9 method is a powerful and economical tool for genome modification and presents an option for analysis of gene expression in not only major model systems, but also in those more diverse species that may not have been amenable to the classic methods of transgenesis. This approach will allow one to generate transgenic lines of snails for future studies. genesis 53:237–244, 2015. © 2014 Wiley Periodicals, Inc.     

CRISPR/Cas9‐mediated mutagenesis of the white gene in the tephritid pest Bactrocera tryoni

The Queensland fruit fly, Bactrocera tryoni (Froggatt), is a polyphagous horticultural pest in Australia that is capable of causing significant damage to more than 100 different host fruits and vegetables. Chemical applications and ecological control strategies, such as the sterile insect technique (SIT), are commonly used to suppress established populations and eradicate invasive outbreaks following migration. The recently published B. tryoni draft genome provides new opportunities to identify candidate genes for targeted genome modification in order to generate advanced genetic strains for management using sterile insect strategies. Here, we demonstrate CRISPR/Cas‐mediated mutagenesis in B. tryoni through generating a series of frame‐shift mutations in the ATP‐dependent binding cassette transporter, white, causing a classic white‐eye phenotype. This work establishes methods for CRISPR/Cas genome editing in tephritids and demonstrates its potential for developing genetic sexing strains which could be used for SIT‐based pest control.     

Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease‐susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9‐targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1^G allele and one copy of the CsLOB1^? allele. The promoter of both alleles contains the effector binding element (EBE_PthA4), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBE_PthA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%–64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBE_PthA4 modifications were identified from 38 mutant plants. Four mutation lines (S2‐5, S2‐6, S2‐12 and S5‐13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2‐6 and S5‐13 lines. Promoter editing of CsLOB1^G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE_PthA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9‐mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker‐resistant citrus cultivars.     

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.     

CRISPR/Cas9‐mediated functional recovery of the recessive rc allele to develop red rice

Red rice contains high levels of proanthocyanidins and anthocyanins, which have been recognized as health‐promoting nutrients. The red coloration of rice grains is controlled by two complementary genes, Rc and Rd. The RcRd genotype produces red pericarp in wild species Oryza rufipogon, whereas most cultivated rice varieties produce white grains resulted from a 14‐bp frame‐shift deletion in the seventh exon of the Rc gene. In the present study, we developed a CRISPR/Cas9‐mediated method to functionally restore the recessive rc allele through reverting the 14‐bp frame‐shift deletion to in‐frame mutations in which the deletions were in multiples of three bases, and successfully converted three elite white pericarp rice varieties into red ones. Rice seeds from T₁ in‐frame Rc lines were measured for proanthocyanidins and anthocyanidins, and high accumulation levels of proanthocyanidins and anthocyanidins were observed in red grains from the mutants. Moreover, there was no significant difference between wild‐type and in‐frame Rc mutants in major agronomic traits, indicating that restoration of Rc function had no negative effect on important agronomic traits in rice. Given that most white pericarp rice varieties are resulted from the 14‐bp deletion in Rc, it is conceivable that our method could be applied to most white pericarp rice varieties and would greatly accelerate the breeding of new red rice varieties with elite agronomic traits. In addition, our study demonstrates an effective approach to restore recessive frame‐shift alleles for crop improvement.     

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off‐target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR‐induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5‐fold in somatic tissues and up to 100‐fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double‐stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on‐target mutagenesis in plants using CRISPR/Cas9.     

Biallelic β‐carotene oxygenase 2 knockout results in yellow fat in sheep via CRISPR/Cas9

The recently emerged CRISPR/Cas9 approach represents an efficient and versatile genome editing tool for producing genetically modified animals. Β‐carotene oxygenase 2 (BCO2) is a key enzyme in the progress of β‐carotene metabolism and is associated with yellow adipose tissue color in sheep. We have recently demonstrated targeted multiplex mutagenesis in sheep and have generated a group of BCO2‐disrupted sheep by zygote injection of the CRISPR/Cas9 components. Here, we show that biallelic modification of BCO2 resulted in yellow fat, compared with the fat color in monoallelic individuals and wild types (snow‐flower white). We subsequently characterized the effects of gene modifications at genetic levels employing sequencing and Western blotting, highlighting the importance of the BCO2 gene for the determination of fat color in sheep. These results indicate that genetic modification via CRISPR/Cas9 holds great potential for validating gene functions as well as for generating desirable phenotypes for economically important traits in livestock.     

CRISPR/Cas9‐mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean

Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens‐mediated transformation. Site‐directed mutations were observed at all targeted sites by DNA sequencing analysis. T1‐generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1‐bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58′, E116°20′). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long‐day and short‐day conditions. We identified some ‘transgene‐clean’ soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These ‘transgene‐clean’ mutants of GmFT2a may provide materials for more in‐depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction.     

Generation of an estrogen receptor beta‐iCre knock‐in mouse

A novel knock‐in mouse that expresses codon‐improved Cre recombinase (iCre) under regulation of the estrogen receptor beta (Esr2) promoter was developed for conditional deletion of genes and for the spatial and/or temporal localization of Esr2 expression. ESR2 is one of two classical nuclear estrogen receptors and displays a spatiotemporal expression pattern and functions that are different from the other estrogen receptor, ESR1. A cassette was constructed that contained iCre, a polyadenylation sequence, and a neomycin selection marker. This construct was used to insert iCre in front of the endogenous start codon of the Esr2 gene of a C57BL/6J embryonic stem cell line via homologous recombination. Resulting Esr2‐iCre mice were bred with ROSA26‐lacZ and Ai9‐RFP reporter mice to visualize cells of functional iCre expression. Strong expression was observed in the ovary, the pituitary, the interstitium of the testes, the head and tail but not body of the epididymis, skeletal muscle, the coagulation gland (anterior prostate), the lung, and the preputial gland. Additional diffuse or patchy expression was observed in the cerebrum, the hypothalamus, the heart, the adrenal gland, the colon, the bladder, and the pads of the paws. Overall, Esr2‐iCre mice will serve as a novel line for conditionally ablating genes in Esr2‐expressing tissues, identifying novel Esr2‐expressing cells, and differentiating the functions of ESR2 and ESR1. genesis 54:38–52, 2016. © 2016 Wiley Periodicals, Inc.     

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.