The extent of multiallelic, co-editing of LIGULELESS1 in highly polyploid sugarcane tunes leaf inclination angle and enables selection of the ideotype for biomass yield.

Sugarcane (Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield. LIGULELESS1 (LG1) is involved in leaf ligule and auricle development in grasses. Here, we report CRISPR/Cas9-mediated co-mutagenesis of up to 40 copies/alleles of the putative LG1 in highly polyploid sugarcane (2n = 100-120, x = 10-12). Next generation sequencing revealed co-editing frequencies of 7.4%-100% of the LG1 reads in 16 of the 78 transgenic lines. LG1 mutations resulted in a tuneable leaf angle phenotype that became more upright as co-editing frequency increased. Three lines with loss of function frequencies of ~12%, ~53% and ~95% of lg1 were selected following a randomized greenhouse trial and grown in replicated, multi-row field plots. The co-edited LG1 mutations were stably maintained in vegetative progenies and the extent of co-editing remained constant in field tested lines L26 and L35. Next generation sequencing confirmed the absence of potential off targets. The leaf inclination angle corresponded to light transmission into the canopy and tiller number. Line L35 displaying loss of function in ~12% of the lg1 NGS reads exhibited an 18% increase in dry biomass yield supported by a 56% decrease in leaf inclination angle, a 31% increase in tiller number, and a 25% increase in internode number. The scalable co-editing of LG1 in highly polyploid sugarcane allows fine-tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield.

CRISPR base editors: genome editing without double-stranded breaks.

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease.

KEY MESSAGE: The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination. The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination.

Genome editing in rice and tomato with a small Un1Cas12f1 nuclease.

The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type-V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1-RRA variant did not perform better than the wild-type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants.

Unsupervised cluster analysis of clinical and ultrasound features reveals unique gout subtypes: Results from the Egyptian College of Rheumatology (ECR).

BACKGROUND: Gout comprises a heterogeneous group of disorders; however, comorbidities have been the focus of most efforts to classify disease subgroups. OBJECTIVES: We applied cluster analysis using musculoskeletal ultrasound (MSUS) combined with clinical and laboratory findings in patients with gout to identify disease phenotypes, and differences across clusters were investigated. PATIENTS AND METHODS: Patients with gout who complied with the ACR/EULAR classification criteria were enrolled in the Egyptian College of Rheumatology (ECR)-MSUS Study Group, a multicenter study. Selected variables included demographic, clinical, and laboratory findings. MSUS scans assessed the bilateral knee and first metatarsophalangeal joints. We performed a K-mean cluster analysis and compared the features of each cluster. RESULTS: 425 patients, 267 (62.8 %) males, mean age 54.2 ± 10.3 years were included. Three distinct clusters were identified. Cluster 1 (n = 138, 32.5 %) has the lowest burden of the disease and a lower frequency of MSUS characteristics than the other clusters. Cluster 2 (n = 140, 32.9 %) was mostly women, with a low rate of urate-lowering treatment (ULT). Cluster 3 (n = 147, 34.6 %) has the highest disease burden and the greatest proportion of comorbidities. Significant MSUS variations were found between clusters 2 and 3: joint effusion (p < 0.0001; highest: cluster 3), power Doppler signal (p < 0.0001; highest: clusters 2), and aggregates of crystal deposition (p < 0.0001; highest: cluster 3). CONCLUSION: Cluster analysis using MSUS findings identified three gout subgroups. People with more MSUS features were more likely to receive ULT. Treatment should be tailored according to the cluster and MSUS features.

Prime editor integrase systems boost targeted DNA insertion and beyond.

Previously developed genome engineering tools cannot efficiently direct site-specific long DNA insertion. Built on the prime editing platform, two recent studies have reported integrase-mediated site-specific long DNA integration in the human genome. These prime editor integrase (PEI) systems will unleash many exciting applications in humans, animals, and plants.

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane.

Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane (x = 10-13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I (MgCh) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents.

Overlapping roles of spliceosomal components SF3B1 and PHF5A in rice splicing regulation.

The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, plays a crucial role in recognizing branch point sequence and facilitates spliceosome assembly and activation. Several chemicals that bind SF3B1 and PHF5A subunits of the SF3B complex inhibit splicing. We recently generated a splicing inhibitor-resistant SF3B1 mutant named SF3B1 GEX1A RESISTANT 4 (SGR4) using CRISPR-mediated directed evolution, whereas splicing inhibitor-resistant mutant of PHF5A (Overexpression-PHF5A GEX1A Resistance, OGR) was generated by expressing an engineered version PHF5A-Y36C. Global analysis of splicing in wild type and these two mutants revealed the role of SF3B1 and PHF5A in splicing regulation. This analysis uncovered a set of genes whose intron retention is regulated by both proteins. Further analysis of these retained introns revealed that they are shorter, have a higher GC content, and contain shorter and weaker polypyrimidine tracts. Furthermore, splicing inhibition increased seedlings sensitivity to salt stress, consistent with emerging roles of splicing regulation in stress responses. In summary, we uncovered the functions of two members of the plant branch point recognition complex. The novel strategies described here should be broadly applicable in elucidating functions of splicing regulators, especially in studying the functions of redundant paralogs in plants.

CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors.

Increasing genetic diversity via directed evolution holds great promise to accelerate trait development and crop improvement. We developed a CRISPR/Cas-based directed evolution platform in plants to evolve the rice (Oryza sativa) SF3B1 spliceosomal protein for resistance to splicing inhibitors. SF3B1 mutant variants, termed SF3B1-GEX1A-Resistant (SGR), confer variable levels of resistance to splicing inhibitors. Studies of the structural basis of the splicing inhibitor binding to SGRs corroborate the resistance phenotype. This directed evolution platform can be used to interrogate and evolve the molecular functions of key biomolecules and to engineer crop traits for improved performance and adaptation under climate change conditions.

Pea early-browning virus-mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis.

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species. Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.

Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule.

The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.

Genome editing: the road of CRISPR/Cas9 from bench to clinic.

Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy.

CRISPR/Cas9-mediated target validation of the splicing inhibitor Pladienolide B.

CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.

Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering.

The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15-39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.