Cas9 Constructs Research Articles

Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants.

Multiplexed Cas9-based genome editing of cotton resulted in reduction of viral load with asymptomatic cotton plants. In depth imaging of proteomic dynamics of resulting CLCuV betasatellite and DNA-A protein was also performed. The notorious cotton leaf curl virus (CLCuV), which is transmitted by the sap-sucking insect whitefly, continuously damages cotton crops. Although the application of various toxins and RNAi has shown some promise, sustained control has not been achieved. Consequently, CRISPR_Cas9 was applied by designing multiplex targets against DNA-A (AC2 and AC3) and betasatellite (βC1) of CLCuV using CRISPR direct and ligating into the destination vector of the plant using gateway ligation method. The successful ligation of targets into the destination vector was confirmed by the amplification of 1049 bp using a primer created from the promoter and target, while restriction digestion using the AflII and Asc1 enzymes determined how compact the plasmid developed and the nucleotide specificity of the plasmid was achieved through Sanger sequencing. PCR confirmed the successful introduction of plasmid into CKC-1 cotton variety. Through Sanger sequencing and correlation with the mRNA expression of DNA-A and betasatellite in genome-edited cotton plants subjected to agroinfiltration of CLCuV infectious clone, the effectiveness of knockout was established. The genome-edited cotton plants demonstrated edited efficacy of 72% for AC2 and AC3 and 90% for the (βC1) through amplicon sequencing, Molecular dynamics (MD) simulations were used to further validate the results. Higher RMSD values for the edited βC1 and AC3 proteins indicated functional loss caused by denaturation. Thus, CRISPR_Cas9 constructs can be rationally designed using high-throughput MD simulation technique. The confidence in using this technology to control plant virus and its vector was determined by the knockout efficiency and the virus inoculation assay.

CRISPR/Cas9 Knock-Out in Primary Neonatal and Adult Cardiomyocytes Reveals Distinct cAMP Dynamics Regulation by Various PDE2A and PDE3A Isoforms.

Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes.

Decorating chromatin for enhanced genome editing using CRISPR-Cas9

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.

Correction of a CD55 mutation to quantify the efficiency of targeted knock-in via flow cytometry.

Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural sciences. However, improvements in the efficiency, precision, and specificity of targeted knock-in are prerequisites to facilitate the practical application of this technology. To improve the efficiency of targeted knock-in, it is necessary to have a molecular system that allows sensitive monitoring of targeted knock-in events with simple procedures. We developed an assay, named CD55 correction assay, with which to monitor CD55 gene correction accomplished by targeted knock-in. To create the reporter clones used in this assay, we initially introduced a 7.7-kb heterozygous deletion covering CD55 exons 2-5, and then incorporated a truncating mutation within exon 4 of the remaining CD55 allele in human cell lines. The resultant reporter clones that lost the CD55 protein on the cell membrane were next transfected with Cas9 constructs along with a donor plasmid carrying wild-type CD55 exon 4. The cells were subsequently stained with fluorescence-labeled CD55 antibody and analyzed by flow cytometry to detect CD55-positive cells. These procedures allow high-throughput, quantitative detection of targeted gene correction events occurring in an endogenous human gene. The current study demonstrated the utility of the CD55 correction assay to sensitively quantify the efficiency of targeted knock-in. When used with the PIGA correction assay, the CD55 correction assay will help accurately determine the efficiency of targeted knock-in, precluding possible experimental biases caused by cell line-specific and locus-specific factors.

A cationic lipid mediated CRISPR/Cas9 technique for the production of stable genome edited citrus plants.

BackgroundThe genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants.ResultsHere, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1.ConclusionsOur results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.

Cas9-Mediated Targeted Mutagenesis in Plants.

Genome engineering technologies enable targeted mutations to be induced at almost any location in plant genomes. In particular, Cas9 nucleases use easily recoded RNA guides to target user-defined sequences and generate double-stranded breaks (DSB) that are then repaired by the cell's endogenous repair mechanisms. Incorrect repair results in mutations at the target. When the targets are in coding sequences, this often results in loss-of-function mutations. In this chapter, we describe a method to rapidly design and assemble RNA-guided Cas9 constructs for plants and test their ability to induce mutations at their intended targets in rapid assays using both Agrobacterium-mediated transient expression and PEG-mediated DNA delivery to protoplasts, the latter of which can be adapted to a wide range of plant species. We describe a PCR-based method for detecting mutagenesis and outline the steps required to segregate the Cas9 transgene from the targeted mutation to enable the production of transgene-free mutated plants. These techniques are amenable to a range of plant species and should accelerate the application of Cas-9-mediated genome engineering for basic plant science as well as crop development.

Genetic and Epigenetic Interplay Within a COLGALT2 Enhancer Associated With Osteoarthritis

ObjectiveThe osteoarthritis (OA)–associated single‐nucleotide polymorphism (SNP) rs11583641 is located in COLGALT2, encoding a posttranslational modifier of collagen. In cartilage, the SNP genotype correlates with DNA methylation in a putative enhancer. This study was undertaken to characterize the mechanistic relationship between rs11583641, the putative enhancer, and COLGALT2 expression using cartilage samples from human patients and a chondrocyte cell model.MethodsNucleic acids were extracted from articular cartilage samples obtained from patients with OA (n = 137). Samples were genotyped, and DNA methylation was quantified at 12 CpGs using pyrosequencing. The putative enhancer was deleted in Tc28a2 chondrocytes using clustered regularly interspaced short palindromic repeat/Cas9, and the impact on nearby gene expression was determined using real‐time quantitative polymerase chain reaction. Targeted modulation of the epigenome using catalytically dead Cas9 (dCas9) constructs fused to DNA methyltransferase 3a or ten–eleven translocase 1 allowed for the investigation of a causal relationship between DNA methylation and enhancer activity.ResultsThe genotype at rs11583641 correlated with DNA methylation at 3 CpGs, and the presence of the OA risk allele, C, corresponded to reduced levels of methylation. Deletion of the enhancer resulted in a 2.7‐fold reduction in COLGALT2 expression. Targeted methylation and demethylation of the CpGs had antagonistic effects on COLGALT2 expression. An allelic imbalance in the expression of COLGALT2 was identified in the cartilage from patients with OA, with relative overexpression of the OA risk allele. Allelic expression ratios correlated with DNA methylation at 4 CpGs.ConclusionCOLGALT2 is a target of OA genetic risk at this locus. The genotype at rs11583641 impacts DNA methylation in a gene enhancer, which, in turn, modulates COLGALT2 expression. COLGALT2 encodes an enzyme that initiates posttranslational glycosylation of collagens and is therefore a compelling OA susceptibility target.

Disruption in the DNA Mismatch Repair Gene MSH2 by CRISPR-Cas9 in Indica Rice Can Create Genetic Variability.

Genetic variation is crucial for crop improvement. We adopted a gene editing approach to create variations in the rice genome by targeting the mutator locus homolog 2 (MSH2), a DNA mismatch repair gene. The hypothesis is that disruption of the MSH2 gene leads to a reduced DNA mismatch repair that creates INDELs, resulting in altered phenotypes. The Indica rice (IR-64) genotype was transformed with a guide RNA targeted to the MSH2 gene using an Agrobacterium-mediated in planta method. Many plants showed integration of Cas9 and gRNA constructs in rice plants. One of the msh2 mutants showed a superior phenotype due to editing and possible INDELs in the whole genome. The stable integration of the transgene and its flanking sequence analysis confirms no disruption of any gene, and the observed phenotype is due to the mutations in the MSH2 gene. Few transgenic plants showed disruption of genes due to T-DNA integration that led to altered phenotypes. The plants with altered phenotypes having more tiller number, early flowering, and robust growth with a high biomass were identified. These genetically reprogrammed rice plants could be a potential resource to create more segregating population or act as donor lines to stabilize the important agronomic traits that may help in a speed breeding process.

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean.

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean.

CRISPR Caspase 9 strategies to explore parkin loss‐of‐function Drosophila development

Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic (DA) neurons selectively degenerate within the substantia nigra pars compacta. Parkin loss‐of‐function Drosophila melanogaster, a model for familial PD, have neurodegeneration in structures homologous to those of the human substantia nigra. Mitochondrial dysfunction and reactive oxygen species (ROS) are implicated in PD‐associated neurodegeneration and in the parkin‐null fly phenotype. In healthy cells, ROS are involved in signaling and are regulated by antioxidant activity. Glutathione (GSH) is a major antioxidant that combats the effects of excess damaging ROS (oxidative stress) and may be dysregulated in parkin mutant Drosophila. Preliminary data from our lab has shown that parkin loss‐of‐function Drosophila (park25/25; Greene et al., 2003) have elevated hydrogen peroxide levels that are sustained as the fly ages, a transient increase in glutathione activity, and increased levels of protein oxidation. Whether oxidative stress persists because of low GSH levels, dysfunction of GSH, or excessive ROS is unclear. Our study aims to address the possibility that low levels of GSH contribute to the parkin loss‐of‐function fly phenotype. To this end, we utilized a modified CRISPR/Cas9 system to overexpress cystathionine beta synthase (CBS), a key, rate‐limiting‐limiting enzyme involved in the production of GSH. To overexpress CBS, we have used a modified Cas9 construct in which the enzymatic function of Cas9 has been deactivated (“dead Cas9” or “dCas9”). Transcriptional activators VPR have been fused to dCas9, resulting in overexpression of CBS. Since parkin–null Drosophila have developmental deficits, we have measured pupariation and eclosion efficiencies in flies overexpressing CBS to shed light on the potential use of this intervention for future studies. Parent flies are placed in a vial and allowed to lay embryos. First and second instar larva are collected and transferred to a secondary vial. The larva are then monitored, and the number of larva that pupate are recorded (pupariation efficiency). The number of flies that eclose (emerge from the pupa case) are also recorded and compared to the pupa count (eclosion efficiency). Additionally, the distance between pupa and the food level is measured to indicate larval mobility. Preliminary data indicate the overexpression of CBS increases pupariation efficiency and decreases pupariation times in the heterozygous parkin mutants. Parkin‐null larva CBS overexpression also appear to have increased climbing. This initial data implicates overexpression of CBS in neuroprotection of the park loss‐of‐function model. Additional experiments on neuron degeneration, mitochondrial functionality, and antioxidant activity can provide further insight into why parkin‐null dopamine neurons are susceptible to degradation. By incorporating Cas9 and dCas9 into our parkin‐null flies, we can modify expression of hundreds of genes in order to elucidate the pathways implicated in oxidative stress in this model.

Mutants in mitochondrial DNA repair genes in Arabidopsis thaliana generated by using CRISPR‐Cas9

The genomes of plant mitochondria exhibit unique mutation rates and structural variability. Compared to their animal counterparts, plant mitochondria have low mutation rates, high recombination rates, and non‐circular genomes. Recent evidence suggests the prevalence of double‐strand break repair (DSBR) in plant mitochondria as one possible cause of these traits. Interestingly, all of the genes required for plant mitochondrial DSBR are located in the nucleus.In order to study DSBR, it is necessary to be able to reliably generate single and double‐mutants for DSBR genes. Using traditional methods, such as T‐DNA insertions, double mutant generation has proven cumbersome due to the occasional difficulty in PCR‐based identification of single mutants, as well as the low map distance between several genes of interest. CRISPR‐induced knockouts allow us to overcome some of these difficulties and generate a greater variety of mutants in a shorter period of time. In this study, we selected five proteins related to the processing of double strand breaks (DSBs) in plant mitochondria for mutagenesis: MSH1, RECA3, RNaseH1, SUV3, and OTP87. We prepared CRISPR Cas9 vectors to produce knockouts for each of these genes in Arabidopsis thaliana. The reliable production of mutants for these 5 genes will allow for in depth study of DSBR in the future. CRISPR Cas9 constructs were designed to produce two guide RNAs for each gene, targeting two different exons in the gene of interest, to give a better chance of successful knockouts, or deletions, between the two sites. These constructs were then transformed into Agrobacterium. Successful transformants were selected using a spectinomycin resistance gene in the construct. We then used a standard floral dip protocol to generate mutant Arabidopsis plants. PCR analysis confirmed Arabidopsis knockout mutations in all five lines.The mutants generated in this study will allow us to investigate several avenues of research on DSBR. We plan to expose plants with knockouts in either one or two DSBR genes to ciprofloxacin, an antibiotic which induces double strand breaks in plant mitochondrial DNA. From there we can observe the mitochondrial genome recombination rates using PCR and Illumina sequencing for each of the mutants.Support or Funding InformationThis work was supported in part by grants from the National Science Foundation to A.C.C. (MCB‐1413152 and MCB‐1933590) and a grant from the UNL UCARE program (26‐0101‐8001‐001)

Spatial and temporal control of gene manipulation in Drosophila via drug-activated Cas9 nucleases

Advances in CRISPR/Cas9 have revolutionized molecular biology and greatly facilitated the ability to manipulate gene function through the creation of precisely engineered mutants. We recently reported a collection of modular gateway-compatible Cas9/gRNA Drosophila lines to interfere with gene expression in a tissue-specific manner, including polytene tissues. However, most current in vivo CRISPR/Cas9 tools cannot temporally control the induction of Cas9 or gRNAs via external stimuli such as RU486. A drug-inducible CRISPR/Cas9 system would allow studying genes at later stages where early lethality is an issue. This would be especially useful when combined with tissue-specific expression of Cas9 or gRNAs, allowing for full spatiotemporal control. Here, we present a RU486-inducible version of Cas9 and also show that a Rapamycin-inducible Cas9, previously used in mammalian cell culture, works in Drosophila as well. Both RU486 and rapamycin-inducible Cas9 work in vivo and in Drosophila cell culture. We also present split Cas9 constructs for rapamycin-dependent gene disruption and activation. These approaches establish drug-inducible and thus temporally controlled CRISPR/Cas9 tools for gene disruption and expression in a living model organism. Our CRISPR/Cas9 vector collection can be easily adapted for any tissue and provides higher fidelity compared to RNAi approaches.

Recruitment of DNA Repair MRN Complex by Intrinsically Disordered Protein Domain Fused to Cas9 Improves Efficiency of CRISPR-Mediated Genome Editing.

CRISPR/Cas9 is a powerful tool for genome editing in cells and organisms. Nevertheless, introducing directed templated changes by homology-directed repair (HDR) requires the cellular DNA repair machinery, such as the MRN complex (Mre11/Rad50/Nbs1). To improve the process, we tailored chimeric constructs of Cas9, in which SpCas9 was fused at its N- or C-terminus to a 126aa intrinsically disordered domain from HSV-1 alkaline nuclease (UL12) that recruits the MRN complex. The chimeric Cas9 constructs were two times more efficient in homology-directed editing of endogenous loci in tissue culture cells. This effect was dependent upon the MRN-recruiting activity of the domain and required lower amounts of the chimeric Cas9 in comparison with unmodified Cas9. The new constructs improved the yield of edited cells when making endogenous point mutations or inserting small tags encoded by oligonucleotide donor DNA (ssODN), and also with larger insertions encoded by plasmid DNA donor templates. Improved editing was achieved with both transfected plasmid-encoded Cas9 constructs as well as recombinant Cas9 protein transfected as ribonucleoprotein complexes. Our strategy was highly efficient in restoring a genetic defect in a cell line, exemplifying the possible implementation of our strategy in gene therapy. These constructs provide a simple approach to improve directed editing.

Nuclease dead Cas9 is a programmable roadblock for DNA replication

Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

Assessment of Cas12a-mediated gene editing efficiency in plants.

SummaryThe CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of −10 to −2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.

A simple and efficient CRISPR/Cas9 platform for induction of single and multiple, heritable mutations in barley (Hordeum vulgare L.)

BackgroundGenome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.ResultsWe developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T0 plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T1 lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T1 plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T1 plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T0 plants.ConclusionsWe demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System.

Ustilaginoidea virens is the causal agent of rice false smut, one of the major fungal diseases of rice. However, there are only limited molecular studies with this important pathogen due to the lack of efficient approaches for generating targeted gene disruption mutants. In this study, we used the CRISPR-Cas9 system to efficiently generate mutants deleted of the USTA ustiloxin and UvSLT2 MAP kinase genes. Three gRNA spacers of USTA, UA01, UA13, and UA21, were expressed with the RNAP III promoter of Gln-tRNA. For all of them, the homologous gene replacement frequency was higher when the Cas9 and gRNA constructs were transformed into U. virens on the same vector than sequentially. UA01, the spacer with the highest on-target score, had the highest knockout frequency of 90%, which was over 200 times higher than that of Agrobacterium tumefaciens-mediated transformation (ATMT) for generating ustA mutants. None of these USTA spacers had predicted off-targets with 1 or 2-nt variations. For predicted off-targets with 3 or 4-nt variations, mutations were not detected in 10 ustA mutants generated with spacer UA13 or UA21, indicating a relatively low frequency of off-target mutations in U. virens. For UvSLT2, the homologous gene replacement frequency was 50% with CRISPR-Cas9, which also was significantly higher than that of ATMT. Whereas ustA mutants had no detectable phenotypes, Uvslt2 mutants were slightly reduced in growth rate and reduced over 70% in conidiation. Deletion of UvSLT2 also increased sensitivity to cell wall stresses but tolerance to hyperosmotic or oxidative stresses. Taken together, our results showed that the CRISPR-Cas9 system can be used as an efficient gene replacement or editing approach in U. virens and the UvSlt2 MAP kinase pathway has a conserved role in cell wall integrity.

A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity

Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA. By incorporating an out-of-frame sequence to the N-terminal of the red fluorescent protein mCherry, we created a visual tool for assessing the indel frequency after cutting with CRISPR Cas9. When a sgRNA-Cas9 construct is co-transfected with a corresponding GEmCherry2 construct, single nucleotide indels can move the GEmCherry2 sequence back in-frame and allow quantification and comparison of the efficiency of different sgRNA target sites by measuring red fluorescence. With this GEmCherry2 assay, we compared four target sites in the safe harbor AAVS1 locus and found significant differences in target site activity. We verified the activity using TIDE, which ranked our target sites in a similar order as the GEmCherry2 system. We also identified an AAV short inverted terminal repeat sequence within the Cas9 construct that, upon removal significantly improved transient transfection and expression in hESCs. Moreover, using GEmCherry2, we designed a sgRNA to target SORCS2 in hESCs and successfully introduced indels into the coding sequence of SORCS2.

Targeted mutagenesis in wheat microspores using CRISPR/Cas9

CRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10–20 µg DNA and a pulsing voltage of 500 V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants.

High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides

Selective retrieval of sequence-verified oligonucleotides (oligos) from next-generation sequencing (NGS) flow cells, termed megacloning, promises accurate and reliable gene synthesis. However, gene assembly requires a complete collection of overlapping sense and nonsense oligos, and megacloning does not typically guarantee the complete production of sequence-verified oligos. Therefore, missing oligos must be provided via repetitive rounds of megacloning, which introduces a bottleneck for scaled-up efforts at gene assembly. Here, we introduce the concept of high-depth tiled oligo design to successfully utilize megacloned oligos for gene synthesis. Using acquired oligos from a single round of the megacloning process, we assembled 72 of 81 target Cas9-coding gene variants. We further validated 62 of these cas9 constructs, and deposited the plasmids to Addgene for subsequent functional characterization by the scientific community. This study demonstrates the utility of using sequence-verified oligos for DNA assembly and provides a practical and reliable optimized method for high-throughput gene synthesis.