gRNA Expression Research Articles

Harnessing tRNA for Processing Ability and Promoter Activity.

Transfer RNA (tRNA) and their associated production and processing machinery can be coopted as a versatile tool for the production of guide RNAs (gRNAs) for Cas9-based genome engineering. Using different tRNA variants enables the production of gRNAs at a variety of steady state levels. Furthermore, engineered tRNAs can be used to process gRNAs from Pol-II transcripts, thus enabling spatial/temporal control of gRNA expression. Here we describe the design, cloning, and testing of tRNA scaffolds for both Pol-III-driven expression of different levels of gRNAs, and for processing gRNAs from Pol-II transcripts.

A Single Cas9-VPR Nuclease for Simultaneous Gene Activation, Repression, and Editing in Saccharomyces cerevisiae.

Combinatorial metabolic engineering has been widely established for the development of efficient microbial cell factories to produce the products of interest by precisely regulating the expression levels of multiple genes simultaneously. Here, we report a novel multifunctional CRISPR system that enables simultaneous gene activation, repression, and editing (CRISPR-ARE) with a single Cas9-VPR protein for combinatorial metabolic engineering applications in Saccharomyces cerevisiae. Via gRNA engineering, we achieved orthogonal transcriptional regulations and genome editing using the nuclease active Cas9-VPR fusion protein, individually or in a combinatorial manner. After establishing a system for stable expression of multiple gRNAs on the same plasmid, we first demonstrated CRISPR-ARE for simultaneous mCherry activation, mVenus repression, and ADE2 disruption in a fluorescence reporter strain. Subsequently, we adopted CRISPR-ARE for simple and fast combinatorial metabolic engineering, which improved the production of α-santalene for 2.66-fold in a single step. Because of its simplicity and modularity, the developed CRISPR-ARE system could be applied for facile multifunctional metabolic engineering of microbial cell factories, particularly for which only a few CRISPR proteins have been characterized.

3069 – GRANULIN, REVEALED BY SINGLE-CELL RNA-SEQUENCING OF DEVELOPING HEMATOPOIETIC CELLS, DRIVES MACROPHAGE AND NEUTROPHIL DIFFERENTIATION

Myeloid cell differentiation leads to the formation of neutrophils and macrophages, which are critical cells for host defenses against infections. In addition, a multitude of other functions beyond their classical inflammatory roles have been assigned to these cells, including wound healing, neurodegenerative diseases and tumorigenesis. Thus, the identification of novel genes that regulate myeloid cell development has undoubtedly the potential to impact diverse areas of human health. Here, we have used deep single-cell RNA-sequencing during the active phase of myelopoiesis in the zebrafish embryo to create a platform for the identification of novel genes required for myeloid cell formation. Using this platform, we have uncovered a role for granulin (GRN) during neutrophil and macrophage differentiation. Leveraging the restricted expression of the zebrafish orthologue grna to the myeloid cell lineage, enabled us to perform loss of function experiments without altering its function in non-hematopoietic cells. With the generation of a zebrafish model of grna deficiency, we reveal that myeloid progenitors are unable to differentiate into neutrophils and macrophages. These grna deficient progenitors are also incapable of triggering a myelopoiesis emergency response, resulting in abnormal wound healing with aberrant collagen depositions. Utilizing CUT&RUN, we identified that Pu.1 directly binds grna enhancers, triggering its expression. Similar to the zebrafish grna, the mammalian granulin is also upregulated in myeloid cells, and controlled by the myeloid transcription factors PU.1 and IRF8. Altogether, our findings uncover the role of Granulin during myeloid cell differentiation, opening a new field of study that will help elucidate the pleiotropic role of this enigmatic protein in inflammation, wound healing, tumor progression and neurodegenerative disease.

Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer.

Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer.

A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription.

To simplify CRISPR/Cas9 genome editing in the industrial filamentous fungus Trichoderma reesei based on in vivo guide RNA (gRNA) transcription. Two putative RNA polymerase III U6 snRNA genes were identified in the genome of T. reesei QM6a by BLASTN using Myceliophthora. thermophila U6 snRNA gene as the template. The regions approximately 500bp upstream of two U6 genes were efficient promoters for the in vivo expression of gRNA. The CRISPR system consisting of Cas9 and in vivo synthesized gRNA under control of the T. reesei U6 snRNA promoters was sufficient to cause a frameshift mutation in the ura5 gene via non-homologous end-joining-mediated events. We report a simple gene editing method using a CRISPR/Cas9-coupled in vivo gRNA transcription system in T. reesei.

A multiplex guide RNA expression system and its efficacy for plant genome engineering

BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

Targeted mutation of GA20ox-2 gene using CRISPR/Cas9 system generated semi-dwarf phenotype in rice

Recently, the engineered CRISPR/Cas9 system has been applied to rapidly and efficiently modify the targeted gene(s) in a wide variety of plants. Recent studies of successful targeted mutagenesis using the CRISPR/Cas9 system with a single gRNA expression in rice plants have been reported. GA20ox-2 is a gene encoding an oxidase enzyme involved in the biosynthesis of gibberellin and linked to sd1 locus. A previous study revealed that mutation of this gene resulted in shorter stature of rice plant due to defects in the gibberellin’s signalling pathway. Here, we studied targeted mutation of OsGA20ox-2 gene in rice using the CRISPR/Cas9 system with the expression of two gRNAs. In this study, we introduced a single plasmid vector of CRISPR/Cas9 system harboring dual gRNAs to modify OsGA20ox-2 gene in a rice model cv. Kitaake via Agrobacterium-mediated transformation. Targeted mutagenesis of OsGA20ox-2 gene using CRISPR/Cas9 generated nine mutated rice lines with a mutation frequency of 90%. Most mutated lines (50%) had mutations in both OsGA20ox-2 gRNA. They resulted in homo-diallelic mutation type with 44 bp deletion, while three lines were heterozygous, one line was homo-diallelic with 2 bp insertion, and one line had no mutation. The K15 mutated rice line was identified as a homozygous two-nucleotide insertion and had the semi-dwarf phenotype, demonstrating that OsGA20ox-2 gene had been disrupted.

Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing.

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system (CRISPR/Cas) has recently become the most powerful tool available for genome engineering in various organisms. With efficient and proper expression of multiple guide RNAs (gRNAs), the CRISPR/Cas system is particularly suitable for multiplex genome editing. During the past several years, different CRISPR/Cas expression strategies, such as two-component transcriptional unit, single transcriptional unit, and bidirectional promoter systems, have been developed to efficiently express gRNAs as well as Cas nucleases. Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes, endogenous RNases, and exogenous endoribonuclease (Csy4). Besides being constitutively and ubiquitously expressed, inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible, tissue-specific, and/or synthetic promoters for specific research purposes. Most recently, the emergence of CRISPR/Cas ribonucleoprotein delivery methods, such as engineered nanoparticles, further revolutionized transgene-free and multiplex genome editing. In this review, we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.

Immediate, multiplexed and sequential genome engineering facilitated by CRISPR/Cas9 in Saccharomyces cerevisiae.

A method called Cas-3P allowing for immediate, multiplexed and sequential genome engineering was developed using one plasmid expressing Cas9 and three marked plasmid backbones (P1, P2 and P3) for guide RNA (gRNA) expression. The three marked gRNA plasmid backbones were recurred in a P1-P2-P3 order for sequential gene targeting, without construction of any additional plasmid and elimination of gRNA plasmid by induction in each round. The efficiency of direct gRNA plasmid curing mediated by Cas-3P was more than 40% in sequential gene targeting. Besides, Cas-3P allowed single-, double- and triple-loci gene targeting with an efficiency of 75%, 36.8% and 8.2% within 3-4days, respectively. Through three sequential rounds of gene targeting within 10 days, S. cerevisiae was optimized for the production of patchoulol by replacing one promoter, overexpressing three genes and disrupting four genes. The work is important for practical application in the cell factory engineering of S. cerevisiae.

Editing of the MYB genes inBrassica napusas a method to increase anthocyanin pigmentation and stress tolerance

Anthocyanin hyperaccumulation is an important agricultural trait, associated with resistance to abiotic stress, pests, phytopathogenic fungi and bacterial diseases.B. napuswith increased anthocyanin pigmentation can be generated by genome editing. Many transcription factors of the MYB family are involved in stress response and anthocyanin biosynthesis. GenesAtMYB60, AtCPCandAtMYBL2are negative regulators of anthocyanin biosynthesis inArabidopsis, therefore the knockout of these genes can result in increased anthocyanin pigmentation. gRNA spacers were synthesized to target the orthologs of these genes, identified inBrassica napus. Resulting genetic constructs were introduced to the plant tissues by agroinfiltration. Transient expression of gRNAs targeting DNA-binding domains of MYB transcription factors along with Cas9 nuclease successfully promoted anthocyanin hyperaccumulation. These genetic constructs can be used for genome editing and production of new colored and stress tolerant varieties of oilseed rape.

Improvement and use of CRISPR/Cas9 to engineer a sperm-marking strain for the invasive fruit pest Drosophila suzukii

BackgroundThe invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and disease vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system.ResultsTo improve genome editing, we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses DsRed ubiquitously as a target platform. In addition, we isolated the Ds_β2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing.ConclusionThe deployment of the endogenous promoters of the D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the β2t gene can be used in developing novel transgenic pest control approaches and the CRISPR/Cas9 system as an additional tool for the modification of previously established transgenes.

CRISPR/Cas9-mediated double knockout of SRPK1 and SRPK2 in a nasopharyngeal carcinoma cell line.

Serine-arginine protein kinase (SRPK) is a regulator of alternative splicing events via phosphorylation of splicing factor proteins. Oncogenic roles of SRPK1 and SRPK2 have been reported in various types of cancer. To date, only SRPK1/2 specific inhibitors and small interfering RNA (siRNA) have been used for halting their function momentarily; however, there is no attempt to generate SRPK1/2 stable knockout cancer cells as a tool to investigate their roles in tumorigenesis. Our objective is therefore to establish a nasopharyngeal carcinoma (NPC) cell line with stable SRPK1 or SRPK2 knockout and SRPK1/2 double knockout as a model to investigate their potential roles in NPC. CNE1 was selected as a representative of NPC cell lines to create single and double knockout of SRPK1/2 proteins. SRPK1/2 KO plasmid with cas9, green fluorescent protein (GFP), and gRNA expression was cotransfected with SRPK1/2 homology-directed repair (HDR) plasmid containing puromycin resistance, red fluorescent protein (RFP), and 5' and 3' arm sequence for homologous recombination to CNE1 cells. The transfected CNE1 cells with GFP and RFP expression were sorted through fluorescence-activated cell sorting for further treatment with puromycin containing medium. This step generated stable single knockout of SRPK1 and SRPK2. The SRPK2 knockout NPC cells were used as a precursor for double knockout generation via transfection with Cre plasmid for excision of inserted material to generate puromycin-sensitive SRPK2 knockout clone. The puromycin-sensitive SRPK2 knockout cells were transfected with SRPK1 KO/HDR plasmid and treated with puromycin-containing medium. The puromycin-resistant cells of SRPK1/2 stable double knockout were expanded, and the corresponding protein expression was confirmed by western immunoblotting analysis. Single and double knockout of SRPK1/2 were established using clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated 9 (Cas9) system in an NPC cell line as a model for investigation of their splicing mechanism in NPC.

CRISPR-Mediated Activation of Endogenous Gene Expression in the Postnatal Heart.

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.

Zebra “Fishing” the Role of Granulin in Hematopoiesis

Granulin (GRN) is a multifunctional protein with anti-inflammatory properties and involved in neurological diseases and tumorigenesis. It contains several cysteine-rich motifs that are unique to this protein, which are conserved from sponges to humans indicating their ancient evolutionary origin. Despite being highly expressed by certain hematopoietic cell lineages, the role that GRN plays in hematopoiesis has reminded elusive. The multifunctional nature of this protein, together with its wide expression in all mammalian cell types has challenged the characterization of its functional role in hematopoiesis due to its effects on other tissues. Therefore, we took advantage of the whole genomic duplication of the zebrafish (Danio rerio) and the high conservation of the cysteine-rich motifs among the zebrafish and human granulins to address this knowledge gap and explore their role in hematopoiesis in vivo. The whole genome duplication that separated teleost fish from mammals resulted in two copies of the granulin gene in the zebrafish (Granulin a and Granulin b, Grna and Grnb respectively). This has allowed us an unprecedented view into the function of this protein in hematopoiesis. We show that like mammals, grnb transcripts are found in all cell types, including hematopoietic cells. In contrast, grna is restricted to hematopoietic cells, including myeloid populations. The distinct cell expression of grna and grnb suggests that, in the zebrafish, grna evolved to specifically function in hematopoiesis, while grnb may have taken on the rest of the biological roles assigned to the mammalian granulin. The zebrafish is an animal model with unique advantages for in vivo studies. Its external development allows us to circumvent the challenges of in utero experimentation required using mammals, permitting the use of non-invasive imaging techniques to study developmental hematopoiesis. In addition, more than 70% of genes identified in the zebrafish are conserved in humans. These, together with its high conservation with the human hematopoietic system has led to a greater understanding and prevention of human hematologic diseases by using this elegant animal model. These unique advantages of the zebrafish, in addition to its genetic amenability allowed us to generate Grna and Grnb single mutants and identify their impact in the hematopoietic system in vivo. While the absence of Grnb did not affect the development of the hematopoietic system, lack of Grna led to decreased differentiation of myeloid precursors into neutrophils and macrophages. Therefore, Grna knockout allowed us to disrupt the hematopoietic function of granulin while keeping unaltered its function in the brain and other non-hematopoietic tissues. Although viable, adult Grna mutants developed kidney marrow (the fish analogous to the mammalian bone marrow) failure, with increased progenitors and decreased mature myeloid cells. Mechanistically, we found that pu.1, the main transcription factor that leads to myeloid differentiation, directly bound grna enhancers, upregulating its expression. We have demonstrated that Grna enhanced myeloid gene expression, and decreased gata1 expression thereby facilitating myeloid differentiation and inhibiting the erythroid genetic program. Finally, we show that these findings in the zebrafish are also conserved in humans. Altogether, we have identified the hematopoietic role of granulin without disturbing its biological functions in other tissues. We have unveiled a powerful and novel master regulator for myeloid differentiation that could potentially be utilized for the treatment of hematological disorders such as neutropenia and leukemia. Disclosures No relevant conflicts of interest to declare.

A Functional Analysis of SNP on Anthracycline-Induced Cardiotoxicity in a Uniform Genetic Background Using CRISPR/Cas9 Mediated Single-Base-Edited iPSCs

[Introduction] Anthracycline-induced cardiotoxicity (ACT) has been a major problem in leukemia therapy, and reported to be associated with several candidates of single nucleotide polymorphisms (SNPs). C242T polymorphism (rs4673) in the cytochrome b-245 alpha chain (CYBA) gene, which encodes superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase light chain subunit, is focused on as one of ACT related SNPs. Several clinical studies indicated that rs4673 T allele increases the risk of ACT, however, it is unclear how rs4673 affects ACT. Here, we established a series of induced pluripotent stem cells (iPSCs) with a genotype of C/C, T/T or C/T of rs4673 in a uniform genetic background by using CRISPR/ Cas9, and evaluated the effect of each SNP genotype on ACT in vitro. [Methods and Results] To edit the SNP site using CRISPR/Cas9, three guide RNAs (gRNAs) were designed to induce double strand break (DSB) near the target site. Cas9 nuclease and gRNA expression vectors were transfected into HEK293T cells using jetPEI (Polyplus-transfection SAS), and the genome editing activity was assessed by T7 endonucleaseⅠ(T7E1) assay to select more efficient gRNA for cleavage near the target site. The indel frequencies of sgRNAs 1,2 and 3 were 29.4%, 25.6%, and 18.6%, respectively. Furthermore, oligo-DNA (100 nt) to edit single-base in the SNP site was prepared. To generate Cas9/gRNA ribonucleoprotein (RNP) complexes, 1 μg of Cas9 protein and 0.2 μg of gRNA molecules were mixed. RNP complexes and 50 pmol of oligo-DNA were electroporated into human iPSC 201B7cells (Riken BioResource Center) using Neon electroporation device (Invitrogen),single-base-edited iPSCs (C/C and T/T from C/T genotype in rs4673) were established by homology-directed repair (HDR). Successful HDR events were verified by Sanger sequencing. These established iPSCs were differentiated into cardiomyocytes using PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific) in vitro. To facilitate nascent mesoderm induction, TBX6 was expressed transiently by a tetracycline-inducible lentiviral vector. Differentiated cardiomyocytes were revealed to be contracting and be positive for NK2 transcription factor related locus 5 (NKX2-5) and troponin T2 (TNNT2) by immunofluorescence staining.In order to evaluate the ACT depending on the difference of single base in rs4673, doxorubicin (DOX)was administered to each edited iPSC-derived cardiomyocyte. NADP+/NADPH before and after administration of DOX (1μM) in C/C cardiomyocytes was 1±0.18 and 0.93±0.14, respectively. On the other hand, it was 1±0.07 and 1.29±0.03 in T/T cardiomyocytes (p<0.05, Welch test). T/T cardiomyocytes activated NADP+/NADPH cycling after administration of DOX, while C/C cardiomyocytes did not. [Conclusion] SNPs are a factor of genetic diversity among individuals, and have been revealed to be associated with various traits and diseases by Genome-Wide Association Study (GWAS). However, the diverse genetic backgrounds often hamper to analyze the influence of each SNP on the trait. In this study, we analyzed the effects of rs4673 on ACT in a uniform genetic background by using CRISPR/Cas9 edited iPSCs. Our study showed that T/T cardiomyocytes activated NADP+/NADPH cycling after administration of DOX. These results suggest that rs4673T allele may increase the risk of ACT. Genome editing mediated single-base-alteration will provide us a novel system for functional analysis of SNPs. Disclosures No relevant conflicts of interest to declare.

Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development.

Whitefly-transmitted begomoviruses cause serious damage to many economically important food, feed, and fiber crops. Numerous vegetable crops are severely affected and chilli leaf curl virus (ChiLCV) is the most dominant and widely distributed begomovirus in chilli (Capsicum annuum) throughout the Indian subcontinent. Recently, CRISPR-Cas9 technology was used as a means to reduce geminivirus replication in infected plants. However, this approach was shown to have certain limitations such as the evolution of escape mutants. In this study, we used a novel, multiplexed guide RNA (gRNA) based CRISPR-Cas9 approach that targets the viral genome at two or more sites simultaneously. This tactic was effective in eliminating the ChiLCV genome without recurrence of functional escape mutants. Six individual gRNA spacer sequences were designed from the ChiLCV genome and in vitro assays confirmed the cleavage behaviour of these spacer sequences. Multiplexed gRNA expression clones, based on combinations of the above-mentioned spacer sequences, were developed. A total of nine-duplex and two-triplex CRISPR-Cas9 constructs were made. The efficacy of these constructs was tested for inhibition of ChiLCV infection in Nicotiana benthamiana. Results indicated that all the constructs caused a significant reduction in viral DNA accumulation. In particular, three constructs (gRNA5+4, gRNA5+2 and gRNA1+2) were most effective in reducing the viral titer and symptoms. T7E1 assay and sequencing of the targeted viral genome did not detect any escape mutants. The multiplexed genome-editing technique could be an effective way to trigger a high level of resistance against begemoviruses. To our knowledge, this is the first report of demonstrating the effectiveness of a multiplexed gRNA-based plant virus genome editing to minimize and eliminate escape mutant formation.

Abstract 372: CRISPR-based Gene Activation for Transcriptional Reprograming of Mammalian Cardiomyocytes

Cardiac remodelling is accompanied by silencing of genes necessary for cardiac homeostasis including Krüppel-like factor 15 ( Klf15 ), an anti-hypertrophic factor. Due to a lack of tools for gene activation, we aim to adapt a CRISPR-based approach for gene expression control for mammalian cardiomyocytes. We generated a transgenic mouse model (TG) expressing an enzymatically inactive, gRNA-programmable Cas9 protein under Myh6 promoter control fused to transcriptional activators (VPR) to drive gene expression. As a proof of concept, we systemically injected mice at postnatal day P4 with AAV9 carrying a triple gRNA expression cassette containing gRNAs targeted to the Klf15 promoter region. Significant Klf15 activation was observed at P10 up to 10-fold compared to controls in three independent TG lines. This Klf15 activation was sufficient to drive target gene expression of Aldh2 and Adhfe1 (controls = WT + saline, WT + AAV9, TG + saline, n ≥ 3 per group, ANOVA and Bonferroni correction). Importantly, at the neonatal stage, the Klf15 promoter is characterized by low H3K27ac presence indicating that endogenous gene activation can enhance transcription from an epigenetically inactive locus. We demonstrated that CRISPR/Cas9 mediated loss of KLF15 expression results in impaired contractile function in a 3D model of engineering heart muscle, similar to the Klf15 -/- mouse heart phenotype. Encouraged by the evolutionary conserved KLF15 -mediated mechanisms, we aimed to generate a human induced pluripotent stem cells, which constitutively express dCas9VPR (hIPSC-dCas9VPR) for gene activation in hIPSC-derived cardiomyocytes to complement the in vivo findings upon disease. We targeted the transgene integration to the AAVS1 site by CRISPR gene editing, confirmed correct integration by PCR followed by sequencing and hIPSC-dCas9VPR were successfully differentiated into beating cardiomyocytes. To this end, we have tested gRNAs targeted to the KLF15 promoter region in HEK293 cells and observed significant KLF15 activation of up to 5.5-fold (n = 3, ANOVA and Bonferroni correction). In summary, we report transgenic mammalian tools for endogenous gene (re-) activation purposes to identify potential therapeutic targets for preventing heart failure progression.

Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes

Aspergillus niger is an important industrial producer of enzymes due to its high capacity for producing exocellular secretory proteins. The CRISPR/Cas9 system has been developed as a genetic manipulation tool in A. niger. However, only the basic functions of the CRISPR/Cas9 system, such as codon optimization of Cas9 nucleases and promoter screening of guide RNA (gRNA) expression, have been developed in A. niger. The CRISPR/Cas9 system for manipulating large genomic fragments and multiple gene knock-ins still needs to be established. Here, we improved the CRISPR/Cas9 homologous direct repair (CRISPR-HDR) tool box based on donor DNAs (dDNAs) and plasmid harboring AMA1 and the pyrG marker, allowing recycling of pyrG and Cas9 components. Furthermore, we used the CRISPR-HDR tool box to knock out the 0 kb (protospacer only), 2 kb, 10 kb and even 50 kb gene fragments. This CRISPR-HDR tool box could also be used to simultaneously knock in multiple genes at the loci of two highly expressed extracellular secreted proteins, glucoamylase A (glaA) and alpha-amylase (amyA, two copies). In our study, two or three copies of glucose oxidase (goxC) were precisely knocked in at the loci of amyA and glaA, resulting in 4-fold increased enzyme activity (869.86 U/mL). This CRISPR-HDR tool box can be easily manipulated, and the AMA1-based plasmid can be easily removed under selective pressure of 5-fluoroorotic acid and uridine.

Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts

BackgroundCRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand.ResultsIn this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3–5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations.ConclusionsFACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.

Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.