Endogenous tRNA Research Articles

Bioorthogonal labeling and profiling of N6-isopentenyladenosine (i6A) modified RNA.

Chemical modifications in RNAs play crucial roles in diversifying their structures and regulating numerous biochemical processes. Since the 1990s, several hydrophobic prenyl-modifications have been discovered in various RNAs. Prenyl groups serve as precursors for terpenes and many other biological molecules. The processes of prenylation in different macromolecules have been extensively studied. We introduce here a novel chemical biology toolkit that not only labels i6A, a prenyl-modified RNA residue, by leveraging the unique reactivity of the prenyl group, but also provides a general strategy to incorporate fluorescence functionalities into RNAs for molecular tracking purposes. Our findings revealed that iodine-mediated cyclization reactions of the prenyl group occur rapidly, transforming i6A from a hydrogen-bond acceptor to a donor. Based on this reactivity, we developed an Iodine-Mediated Cyclization and Reverse Transcription (IMCRT) tRNA-seq method, which can profile all nine endogenous tRNAs containing i6A residues in Saccharomyces cerevisiae with single-base resolution. Furthermore, under stress conditions, we observed a decline in i6A levels in budding yeast, accompanied by significant decrease of mutation rate at A37 position. Thus, the IMCRT tRNA-seq method not only permits semi-quantification of i6A levels in tRNAs but also holds potential for transcriptome-wide detection and analysis of various RNA species containing i6A modifications.

An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production.

The CRISPR/Cas9 technology is being employed as a convenient tool for genetic engineering of the industrially important filamentous fungus Trichoderma reesei. However, multiplex gene editing is still constrained by the sgRNA processing capability, hindering strain improvement of T. reesei for the production of lignocellulose-degrading enzymes and recombinant proteins. Here, a CRISPR/Cas9 system based on a multiple sgRNA processing platform was established for genome editing in T. reesei. The platform contains the arrayed tRNA-sgRNA architecture directed by a 5S rRNA promoter to generate multiple sgRNAs from a single transcript by the endogenous tRNA processing system. With this system, two sgRNAs targeting cre1 (encoding the carbon catabolite repressor 1) were designed and the precise deletion of cre1 was obtained, demonstrating the efficiency of sgRNAs processing in the tRNA-sgRNA architecture. Moreover, overexpression of xyr1-A824V (encoding a key activator for cellulase/xylanase expression) at the ace1 (encoding a repressor for cellulase/xylanase expression) locus was achieved by designing two sgRNAs targeting ace1 in the system, resulting in the significantly enhanced production of cellulase (up to 1- and 18-fold on the Avicel and glucose, respectively) and xylanase (up to 11- and 41-fold on the Avicel and glucose, respectively). Furthermore, heterologous expression of the glucose oxidase gene from Aspergillus niger ATCC 9029 at the cbh1 locus with the simultaneous deletion of cbh1 and cbh2 (two cellobiohydrolase coding genes) by designing four sgRNAs targeting cbh1 and cbh2 in the system was acquired, and the glucose oxidase produced by T. reesei reached 43.77U/mL. Besides, it was found the ER-associated protein degradation (ERAD) level was decreased in the glucose oxidase-producing strain, which was likely due to the reduction of secretion pressure by deletion of the major endogenous cellulase-encoding genes. The tRNA-gRNA array-based CRISPR-Cas9 editing system was successfully developed in T. reesei. This system would accelerate engineering of T. reesei for high-level production of enzymes including lignocellulose-degrading enzymes and other recombinant enzymes. Furthermore, it would expand the CRISPR toolbox for fungal genome editing and synthetic biology.

GANT-61 induces cell cycle resting and autophagy by down-regulating RNAP III signal pathway and tRNA-Gly-CCC synthesis to combate chondrosarcoma.

Chondrosarcoma is ineffective for conventional radiotherapy and chemotherapy with a poor prognosis. Hedgehog (Hh) signal pathway plays a crucial role in tumor growth and progression, which is constitutive activated in chondrosarcoma. GLI transcription factors as targets for new drugs or interference technology for the treatment of chondrosarcoma are of great significance. In this study, we indicated that the Hedgehog-GLI1 signal pathway is activated in chondrosarcoma, which further enhances the RNAP III signal pathway to mediate endogenous tRNA fragments synthesis. Downstream oncology functions of endogenous tRNA fragments, such as "cell cycle" and "death receptor binding", are involved in malignant chondrosarcoma. The GANT-61, as an inhibitor of GLI1, could inhibit chondrosarcoma tumor growth effectively by inhibiting the RNAP III signal pathway and tRNA-Gly-CCC synthesis in vivo. Induced G2/M cell cycle resting, apoptosis, and autophagy were the main mechanisms for the inhibitory effect of GANT-61 on chondrosarcoma, which correspond with the above-described downstream oncology functions of endogenous tRNA fragments. We also identified the molecular mechanism by which GANT-61-induced autophagy is involved in ULK1 expression and MAPK signaling pathway. Thus, GANT-61 will be an ideal and promising strategy for combating chondrosarcoma.

Multiplexed genome engineering for porcine fetal fibroblasts with gRNA–tRNA arrays based on CRISPR/Cas9

Multiplex gene modifications are highly required for various fields of porcine research. In many species, the CRISPR/Cas9 system has been widely applied for genomic editing and provides a potential tool for introducing multiplex genome mutations simultaneously. Here, we present a CRISPR–Cas9 gRNA–tRNA array (GTR–CRISPR) for multiplexed engineering of porcine fetal fibroblasts (PFFs). We successfully produced multiple sgRNAs using only one Pol III promoter by taking advantage of the endogenous tRNA processing mechanism in porcine cells. Using an all-in-one construct carrying GTR and Cas9, we disrupted the IGFBP3, MSTN, MC4R, and SOCS2 genes in multiple codon regions in one PFF cell simultaneously. This technique allows the simultaneous disruption of four genes with 5.5% efficiency. As a result, this approach may effectively target multiple genes at the same time, making it a powerful tool for establishing multiple genes mutant cells in pigs.

Impact of queuosine modification of endogenous E. coli tRNAs on sense codon reassignment.

The extent to which alteration of endogenous tRNA modifications may be exploited to improve genetic code expansion efforts has not been broadly investigated. Modifications of tRNAs are strongly conserved evolutionarily, but the vast majority of E. coli tRNA modifications are not essential. We identified queuosine (Q), a non-essential, hypermodified guanosine nucleoside found in position 34 of the anticodons of four E. coli tRNAs as a modification that could potentially be utilized to improve sense codon reassignment. One suggested purpose of queuosine modification is to reduce the preference of tRNAs with guanosine (G) at position 34 of the anticodon for decoding cytosine (C) ending codons over uridine (U) ending codons. We hypothesized that introduced orthogonal translation machinery with adenine (A) at position 34 would reassign U-ending codons more effectively in queuosine-deficient E. coli. We evaluated the ability of introduced orthogonal tRNAs with AUN anticodons to reassign three of the four U-ending codons normally decoded by Q34 endogenous tRNAs: histidine CAU, asparagine AAU, and aspartic acid GAU in the presence and absence of queuosine modification. We found that sense codon reassignment efficiencies in queuosine-deficient strains are slightly improved at Asn AAU, equivalent at His CAU, and less efficient at Asp GAU codons. Utilization of orthogonal pair-directed sense codon reassignment to evaluate competition events that do not occur in the standard genetic code suggests that tRNAs with inosine (I, 6-deaminated A) at position 34 compete much more favorably against G34 tRNAs than Q34 tRNAs. Continued evaluation of sense codon reassignment following targeted alterations to endogenous tRNA modifications has the potential to shed new light on the web of interactions that combine to preserve the fidelity of the genetic code as well as identify opportunities for exploitation in systems with expanded genetic codes.

Queuosine salvage in fission yeast by Qng1-mediated hydrolysis to queuine

Queuosine (Q) is a hypermodified 7-deaza-guanosine nucleoside that is found at position 34, also known as the wobble position, of tRNAs with a GUN anticodon, and Q ensures faithful translation of the respective C- and U-ending codons. While Q is present in tRNAs in most eukaryotes, only bacteria can synthesize it denovo. In contrast, eukaryotes rely on external sources like their food and the gut microbiome in order to Q-modify their tRNAs, and Q therefore can be regarded as a micronutrient. The eukaryotic tRNA guanine transglycosylase (eTGT) uses the base queuine (q) as a substrate to replace G34 by Q in the tRNAs. Eukaryotic cells can uptake both q and Q, raising the question how the Q nucleoside is converted to q for incorporation into the tRNAs. Here, we identified Qng1 (also termed Duf2419) as a queuosine nucleoside glycosylase in Schizosaccharomyces pombe. S. pombe cells with a deletion of qng1+ contained Q-modified tRNAs only when cultured in the presence of the nucleobase q, but not with the nucleoside Q, indicating that the cells are proficient at q incorporation, but not in Q hydrolysis. Furthermore, purified recombinant Qng1 hydrolyzed Q to q in vitro. Qng1 displays homology to DNA glycosylases and has orthologs across eukaryotes, including flies, mice and humans. Qng1 therefore plays an essential role in allowing eukaryotic cells to salvage Q from bacterial sources and to recycle Q from endogenous tRNAs.

Efficient CRISPR/Cas9 system based on autonomously replicating plasmid with an AMA1 sequence and precisely targeted gene deletion in the edible fungus, Cordyceps militaris.

Cordyceps militaris is a popular edible fungus with important economic value worldwide. In this study, an efficient CRISPR/Cas9 genome‐editing system based on an autonomously replicating plasmid with an AMA1 sequence was constructed. Further, a precisely targeted gene deletion via homology‐directed repair was effectively introduced in C. militaris. Gene editing was successful, with efficiencies of 55.1% and 89% for Cmwc‐1 and Cmvvd, respectively. Precisely targeted gene deletion was achieved at an efficiency of 73.9% by a single guide RNA supplementation with donor DNAs. Double genes, Cmwc‐1 and Cmvvd, were edited simultaneously with an efficiency of 10%. Plasmid loss was observed under non‐selective culture conditions, which could permit recycling of the selectable marker and avoid the adverse effects of the CRISPR/Cas9 system on the fungus, which is beneficial for the generation of new cultivars. RNA Pol III promoters, endogenous tRNA Pro of C. militaris, and chimeric AfU6‐tRNA Gly can be used to improve the efficiency. Polyethylene glycol‐mediated protoplast transformation was markedly more efficient than Agrobacterium tumefaciens‐mediated transformation of C. militaris. To our knowledge, this is the first description of genome editing and precisely targeted gene deletion in mushrooms based on AMA1 plasmids. Our findings will enable the modification of multiple genes in both functional genomics research and strain breeding.

Multiplex base- and prime-editing with drive-and-process CRISPR arrays

Current base- and prime-editing technologies lack efficient strategies to edit multiple genomic loci simultaneously, limiting their applications in complex genomics and polygenic diseases. Here, we describe drive-and-process (DAP) CRISPR array architectures for multiplex base-editing (MBE) and multiplex prime-editing (MPE) in human cells. We leverage tRNA as the RNA polymerase III promoter to drive the expression of tandemly assembled tRNA-guide RNA (gRNA) arrays, of which the individual gRNAs are released by the cellular endogenous tRNA processing machinery. We engineer a 75-nt human cysteine tRNA (hCtRNA) for the DAP array, achieving up to 31-loci MBE and up to 3-loci MPE. By applying MBE or MPE elements for deliveries via adeno-associated virus (AAV) and lentivirus, we demonstrate simultaneous editing of multiple disease-relevant genomic loci. Our work streamlines the expression and processing of gRNAs on a single array and establishes efficient MBE and MPE strategies for biomedical research and therapeutic applications.

Protonation‐Dependent Sequencing of 5‐Formylcytidine in RNA

Chemical modification of cytidine in non‐coding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remains unknown due to a lack of sensitive site‐specific sequencing technologies. Here we report a protonation‐dependent sequencing reaction for detection of 5‐formylcytidine (5fC) and 5‐carboxycytidine (5caC) in RNA. First, we evaluate how protonation combined with electron withdrawing substituents alters the molecular orbital energies and reduction of modified cytidine nucleosides, highlighting 5fC and 5caC as reactive species. Next, we apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human tRNA. Finally, we demonstrate the utility of our method to characterize a patient‐derived model of 5fC‐deficiency, where it enables facile monitoring of both pathogenic loss and exogenous rescue of NSUN3‐dependent 5fC within the wobble base of human mitochondrial tRNAMet. These studies showcase the ability of protonation to enhance the reactivity and sensitive detection of 5fC in RNA, and provide a molecular foundation for applying optimized sequencing reactions to better understand the role of oxidized RNA cytidine nucleobases in disease.

AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice.

Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV)is the most successful gene delivery vehicle for in vivo applications1-3. However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene4-6. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.

In lysate RNA digestion provides insights into the angiogenin’s specificity towards transfer RNAs

ABSTRACT Under adverse conditions, tRNAs are processed into fragments called tRNA-derived stress-induced RNAs (tiRNAs) by stress-responsive ribonucleases (RNases) such as angiogenin (ANG). Recent studies have reported several biological functions of synthetic tiRNAs lacking post-transcriptional modifications found on endogenous tiRNAs. Here we describe a simple and reproducible method to efficiently isolate ANG-cleaved tiRNAs from endogenous tRNAs. Using this in vitro method, more than 50% of mature tRNAs are cleaved into tiRNAs which can be enriched using complementary oligonucleotides. Using this method, the yield of isolated endogenous 5ʹ-tiRNAGly-GCC was increased about fivefold compared to when tiRNAs were obtained by cellular treatment of ANG. Although the non-specific ribonuclease activity of ANG is much lower than that of RNase A, we show that ANG cleaves physiologically folded tRNAs as efficiently as bovine RNase A. These results suggest that ANG is highly specialized to cleave physiologically folded tRNAs. Our method will greatly facilitate the analysis of endogenous tiRNAs to elucidate the physiological functions of ANG.

Transfer RNA-Derived Fragments, the Underappreciated Regulatory Small RNAs in Microbial Pathogenesis.

In eukaryotic organisms, transfer RNA (tRNA)-derived fragments have diverse biological functions. Considering the conserved sequences of tRNAs, it is not surprising that endogenous tRNA fragments in bacteria also play important regulatory roles. Recent studies have shown that microbes secrete extracellular vesicles (EVs) containing tRNA fragments and that the EVs deliver tRNA fragments to eukaryotic hosts where they regulate gene expression. Here, we review the literature describing microbial tRNA fragment biogenesis and how the fragments secreted in microbial EVs suppress the host immune response, thereby facilitating chronic infection. Also, we discuss knowledge gaps and research challenges for understanding the pathogenic roles of microbial tRNA fragments in regulating the host response to infection.

Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines.

Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.

TRNA-derived fragments tRFGlnCTG induced by arterial injury promote vascular smooth muscle cell proliferation

tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs) are originated from the specific cleavage of endogenous tRNAs or their precursors and regulate gene expression when the cells are in stressful circumstances. Here, we replicated the rat common carotid artery (CCA) intimal hyperplasia model and investigated the expression of tRFs/tiRNAs in the artery. The normal and the balloon-injured rat CCAs were subjected to small RNA sequencing, and then the differentially expressed tRFs/tiRNAs were identified and analyzed. The expression profiles of tRFs/tiRNAs in the healthy and injured CCAs were remarkably different. tRNAGlnCTG-derived fragments (tRFGlnCTG) were found to be overexpressed with a high abundance in the injured CCA. In in vitro experiments, the synthetic tRFGlnCTG mimetics elevated the proliferation and migration of rat vascular smooth muscle cells (VSMCs). Through bioinformatics analysis and an overexpression experiment, tRFGlnCTG was found to negatively regulate the expression of FAS cell surface death receptor (FAS). This study revealed that tRFGlnCTG is a crucial regulator in promoting VSMC proliferation. The investigation of the roles of tRFs/tiRNAs is of significance for understanding the mechanism, diagnosis, and treatment of intimal hyperplasia.

The Influence of Competing tRNA Abundance on Translation: Quantifying the Efficiency of Sense Codon Reassignment at Rarely Used Codons.

A quantitative understanding of how system composition and molecular properties conspire to determine the fidelity of translation is lacking. Our strategy directs an orthogonal tRNA to directly compete against endogenous tRNAs to decode individual targeted codons in a GFP reporter. Sets of directed sense codon reassignment measurements allow the isolation of particular factors contributing to translational fidelity. In this work, we isolated the effect of tRNA concentration on translational fidelity by evaluating reassignment of the 15 least commonly employed E. coli sense codons. Eight of the rarely used codons are reassigned with greater than 20 % efficiency. Both tRNA abundance and codon demand moderately inversely correlate with reassignment efficiency. Furthermore, the reassignment of rarely used codons does not appear to confer a fitness advantage relative to reassignment of other codons. These direct competition experiments also map potential targets for genetic code expansion. The isoleucine AUA codon is particularly attractive for the incorporation of noncanonical amino acids, with a nonoptimized reassignment efficiency of nearly 70 %.

Divulging the Enigma of Epitranscriptome in Plasmodium falciparum

In spite of prime posttranscriptional modifications, eukaryotic RNA is subjected to internal epitranscriptomic marks. Thus, these changes influence the rate of transcription which regulates the translational plasticity. However, RNA methylome and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, for the foremost a piece of our work demonstrated the cytosine methylation in the tRNA Asp at the C38 position of Plasmodium falciparum, which is accomplished by the tRNA aspartic acid methyl transferase1 (TRDMT1), a conserved homologue of DNA methyltransferase DNMT2. Through RNA bisulfite sequencing, we mapped the 5‐methylcytosines in endogenous tRNA Asp, followed by in‐vitro characterisation of TRDMT1. Accordingly, P. falciparum proteome divulged the significant role of polyaspartic acid repeat‐containing proteins and its abundance. Those proteins may play astonishing roles in host‐pathogen interactions. Further studies focus on the generation of TRDMT1 knock out parasites which may aid us to find out the critical role of TRDMT1 mediated C38 methylation of aspartic acid tRNA. In particular, translational regulation of essential proteins which could modulate the malaria parasite pathogenesis. On the other hand, the significant genes in P. falciparum show discrepancy in mRNA expression and its cognate protein, through which ‘time shift’ is displayed. Moreover, P. falciparum exhibits decelerated ring stage as a distinctive policy for drug resistance. In recent years, it is evidently proven that the epigenetic modifications are existed in eukaryotic mRNA, especially methylation at the nucleotide level and is the reason for time shift.In support, our preliminary data's on P. falciparum RNA implied the methylated nucleotides. We anticipate that parasites may modulate the translation process at the transcription level through methylation that could be installed by a multicomponent methyltransferase complex and follow‐up by the RNA binding proteins. Thus so, parasite might adopt the ‘just‐in‐time’ transcription and translation strategy for its emergence that are controlled by epigenetic machinery. Consequently, further mining of RNA methylome in parasite could be insightful about the host pathogen interaction. Hence, by cracking the epitrancriptome of P. falciparum, the underlying mechanisms of dynamic gene regulation of the parasite pathogenesis could be explored.Support or Funding InformationDepartment of science and technology, Government of India.Department of Biotechnology, Government of India.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Genetic Code is Read by an Idiosyncratic and Highly-Connected Decoding Network

The relationship between the synonymous codons in the genome and the isoacceptor tRNAs that decode them defines how the genetic code is read. Deciphering this relationship in vivo is considered challenging because decoding with distinct isoacceptors yields the same primary protein sequence. We solve this problem by creating chimeras between the anticodon stem loop (ASL, the primary determinant of decoding properties) of endogenous tRNAs and the body of an orthogonal tRNA. We use these chimeras to tag the proteome with unnatural amino acids in response to the codons each ASL decodes. By identifying the sites of proteome tagging and quantitatively comparing the extent of tagging by each ASL in response to each codon we profile tRNA usage for nearly 50,000 decoding events in the E. coli genome. We demonstrate that for the amino acids that are encoded entirely within a family codon box, each isoacceptor can read all four codons in the box; this reveals third base decoding that is not predicted by the extended wobble rules. Moreover for amino acids which are encoded by six codons, we reveal that the isoacceptors believed to only decode codons within a family codon box also decode the other two codons for the same amino acid in a distinct box via ‘cross box decoding’; this demonstrates that non-canonical base pairing is used to decode the first and/or second position of certain codon anticodon interactions. Thus, in contrast to the standard view, Watson Crick base pairs are not required at the first and second position of codon anticodon interactions. Finally we reveal that the cell preferentially uses different isoacceptors to decode different occurrences of the same triplet codon sequence at different positions in the genome.

CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system

Cabbage (Brassica oleracea var. capitata) is a biennial plant with strong self-incompatibility and an obligate requirement for prolonged vernalization by exposure to low temperatures to induce flowering. These characteristics significantly increase the difficulty of exploiting novel germplasm induced by physical or chemical mutagens. In this study, we report a CRISPR/Cas9 gene-editing system based on endogenous tRNA processing to induce high efficiency and inheritable mutagenesis in cabbage. Using the phytoene desaturase gene BoPDS, the S-receptor kinase gene BoSRK, and the male-sterility-associated gene BoMS1 as the target genes, multisite and multiple gene mutations were achieved using a construct with tandemly arrayed tRNA-sgRNA architecture to express multiple sgRNAs. The BoSRK3 gene mutation suppressed self-incompatibility completely, converting the self-incompatible line into a self-compatible line. In addition, the BoMS1 gene mutation produced a completely male-sterile mutant, which was highly cross compatible with its nonmutant isoline at the flowering stage as a result of a simultaneous BoSRK3 gene mutation, enabling the economic propagation of the male-sterile line through bee-mediated cross-pollination. Interestingly, higher site mutation efficiency was detected when a guide sequence was inserted into a location in the tandemly arrayed tRNA-sgRNA architecture that was distal from the upstream Pol III promoter. In addition, mutation sites were also detected in the paralogous genes of the BoPDS and BoSRK genes that had fully consistent sequences or base mismatches but beyond the “seed” region in the spacer sequence compared with the target sgRNAs. Collectively, our results demonstrate that the CRISPR/Cas9 system, coupled with an endogenous tRNA-processing system, is an efficient tool to improve cabbage traits.

Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing.

Summary CRISPR‐Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species‐specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first‐generation single transcript unit (STU)‐Cas9 system, STU‐Cas9‐RZ, which is based on hammerhead ribozyme for processing single guide RNAs (sgRNAs). In this study, we developed two new STU‐Cas9 systems and one STU‐Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU‐Cas9 2.0 systems process the sgRNAs with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU‐Cas9‐Csy4 and STU‐Cas9‐tRNA systems showed more robust genome editing efficiencies than our first‐generation STU‐Cas9‐RZ system and the conventional mixed dual promoter system. We further applied the STU‐Cas9‐tRNA system to compare two C to T base editing systems based on rAPOBEC1 and PmCDA1 cytidine deaminases. The results suggest STU‐based PmCDA1 base editor system is highly efficient in rice. The STU‐Cas12a system, based on Cas12a’ self‐processing of a CRISPR RNA (crRNA) array, was also developed and demonstrated for expression of a single crRNA and four crRNAs. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.

A Multiplexed CRISPR/Cas9 Editing System Based on the Endogenous tRNA Processing.

The CRISPR-Cas9 system has become a powerful and popular tool for genome editing due to its efficiency and simplicity. Multiplex genome editing is an important feature of the CRISPR-Cas9 system and requires simultaneous expression of multiple guide RNAs (gRNAs). Here we describe a general method to efficiently produce many gRNAs from a single gene transcript based on the endogenous tRNA-processing system. A step-by-step protocol is provided for the design and construction of the polycistronic tRNA-gRNA (PTG) gene. The PTG method has been demonstrated to be highly efficient for multiplex genome editing in various plant, animal, and microbial species.