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.     

Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9

The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self‐infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar ‘Alamo’ switchgrass. We first developed a transient assay by which a non‐functional green‐fluorescent protein gene containing a 1‐bp frameshift insertion in its 5′ coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium‐mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9‐induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1‐bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding.     

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.     

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen.     

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.     

Chromosomal deletions mediated by CRISPR/Cas9 in Helicoverpa armigera

Helicoverpa armigera, cotton bollworm, is one of the most disastrous pests worldwide, threatening various food and economic crops. Functional genomic tools may provide efficient approaches for its management. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, dependent on a single guide RNA (sgRNA), has been used to induce indels for targeted mutagenesis in cotton bollworm. However, genomic deletions may be more desirable to disrupt the function of noncoding genes or regulatory sequences. By injecting two sgRNAs with Cas9 protein targeting different exons, we obtained predictable genomic deletions of several hundred bases. We achieved this type of modification with different combinations of sgRNA pairs, including HaCad and HaABCC2. Our finding indicated that CRISPR/Cas9 can be used as an efficient tool to engineer genomes with chromosomal deletion in H. armigera.     

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.     

Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana

Engineered nucleases can be used to induce site‐specific double‐strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error‐prone non‐homologous end‐joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single‐strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ‐mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease‐induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ‐mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740‐fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I–SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB‐inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR‐mediated gene targeting systems but also by the combined action of two nickases as DSB‐inducing agents excluding off‐target effects in homologous genomic regions.     

CRISPR/Cas9-mediated targeted T-DNA integration in rice

Key message

Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome.

Abstract

Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50?80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.

     

CRISPR/Cas9‐induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock‐in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near‐maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.     

CRISPR/Cas9: A new tool for the study and control of helminth parasites

Recent reports of CRISPR/Cas9 genome editing in parasitic helminths open up new avenues for research on these dangerous pathogens. However, the complex morphology and life cycles inherent to these parasites present obstacles for the efficient application of CRISPR/Cas9‐targeted mutagenesis. This is especially true with the trematode flukes where only modest levels of gene mutation efficiency have been achieved. Current major challenges in the application of CRISPR/Cas9 for study of parasitic worms thus lie in enhancing gene mutation efficiency and overcoming issues involved in host passage so that mutated parasites survive. Strategies developed for CRISPR/Cas9 studies on Caenorhabditis elegans, protozoa and mammalian cells, including novel delivery methods, the choice of selectable markers, and refining mutation precision represent novel tactics whereby these impediments can be overcome. Furthermore, employing CRISPR/Cas9‐mediated gene drive to interfere with vector transmission represents a novel approach for the control of parasitic worms that is worthy of further exploration.     

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F₁. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species.     

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

     

Generation of a multiplex mutagenesis population via pooled CRISPR‐Cas9 in soya bean

The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR‐Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR‐Cas9 as a mutant screening tool. Here, we report a pooled CRISPR‐Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR‐Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1‐1/1‐2/1‐3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.     

CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity

CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases.