Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.

BACKGROUND: The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency. METHOD: Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies. RESULT: We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration. CONCLUSION: Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.

Improved self-cleaving precipitation tags for efficient column free bioseparations.

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.

In situ Generation of RNA Complexes for Synthetic Molecular Strand-Displacement Circuits in Autonomous Systems.

Synthetic molecular circuits implementing DNA or RNA strand-displacement reactions can be used to build complex systems such as molecular computers and feedback control systems. Despite recent advances, application of nucleic acid-based circuits in vivo remains challenging due to a lack of efficient methods to produce their essential components, namely, multistranded complexes known as gates, in situ, i.e., in living cells or other autonomous systems. Here, we propose the use of naturally occurring self-cleaving ribozymes to cut a single-stranded RNA transcript into a gate complex of shorter strands, thereby opening new possibilities for the autonomous and continuous production of RNA strands in a stoichiometrically and structurally controlled way.

Strategy for generation of replication-competent recombinant rotaviruses expressing multiple foreign genes.

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

Co-expression of self-cleaved multiple proteins derived from Porcine Reproductive and Respiratory Syndrome Virus by bi-cistronic and tri-cistronic DNA vaccines.

Porcine Reproductive and Respiratory Syndrome caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains one of the important diseases in swine industry. A vaccine that is safe, effective and also elicit broad immune response against multiple antigens is desirable. In this study, we developed multi-cistronic DNA vaccines capable of co-expressing multiple structural proteins derived from PRRSV. To preserve the structure and function of each antigen protein, we employed self-cleaving 2A peptides to mediate separation of multiple proteins expressed by multi-cistronic genes. Six bi-cistronic genes encoding PRRSV GP5 and M proteins were generated, by which each construct contains different 2A sequences derived from Foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) either with or without furin cleavage site (Fu). Vectored by the mammalian expression plasmid pTH, all six bi-cistronic genes co-expressed the proteins GP5 and M at comparable level. Importantly, all six types of 2A sequences could mediate a complete self-cleavage of the GP5 and M. We next generated tri-cistronic DNA vaccines co-expressing the PRRSV proteins GP5, M and N. All homologous and heterologous combinations of P2A and F2A in tri-cistronic genes yielded a complete self-cleavage of the GP5, M and N proteins. Our study reports a success in co-expression of multiple PRRSV structural proteins in discrete form from a single vaccine and confirms feasibility of developing one single vaccine that provides broad immune responses against PRRSV.

Biochemical analysis of cleavage and ligation activities of the pistol ribozyme from Paenibacillus polymyxa.

Nine distinct classes of self-cleaving ribozymes are known to date, of which the pistol ribozyme class was discovered only 5 years ago. Self-cleaving ribozymes are able to cleave their own phosphodiester backbone at a specific site with rates much higher than those of spontaneous RNA degradation. Our study focuses on a bioinformatically predicted pistol ribozyme from the bacterium Paenibacillus polymyxa. We provide a biochemical characterization of this ribozyme, which includes an investigation of the effect of various metal ions on ribozyme cleavage and a kinetic analysis of ribozyme activity under increasing Mg2+ concentrations and pH. Based on the obtained results, we discuss a possible catalytic role of divalent metal ions. Moreover, we investigated the ligation activity of the P. polymyxa pistol ribozyme - an aspect that has not been previously analysed for this ribozyme class. We determined that the P. polymyxa pistol ribozyme is almost fully cleaved at equilibrium with the ligation rate constant being nearly 30-fold lower than the cleavage rate constant. In summary, we have characterized an additional representative of this recently discovered ribozyme class isolated from P. polymyxa. We expect that our biochemical characterization of a pistol representative in a cultivatable, genetically tractable organism will support our future investigation of the biological roles of this ribozyme class in bacteria.

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins.

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH).

Optimization of a 2A self-cleaving peptide-based multigene expression system for efficient expression of upstream and downstream genes in silkworm.

The multigene expression system is highly attractive to co-express multiple genes or multi-subunit complex-based genes for their functional studies, and in gene therapy and visual tracking of expressed proteins. However, the current multiple gene co-expression strategies usually suffer from severe inefficiency and unbalanced expression of multiple genes. Here, we report on an improved 2A self-cleaving peptide (2A)-based multigene expression system (2A-MGES), by introducing an optimized Kozak region (Ck) and altering the gene arrangement, both of which contributed to the efficient expression of two fluorescent protein genes in silkworm. By co-expressing DsRed and EGFP genes in insect cells and silkworms, the potent Ck was first found to improve the translation efficiency of downstream genes, and the expression of the flanking genes of 2A were improved by altering the gene arrangement in 2A-MGES. Moreover, we showed that combining Ck and an optimized gene arrangement in 2A-MGES could synergistically improve the expression of genes in the cell. Further, these two flanking genes, regulated by modified 2A-MGES, were further co-expressed in the middle silk gland and secreted into the cocoon, and both achieved efficient expression in the transgenic silkworms and their cocoons. These results suggested that the modified Ck-2A-MGES will be a potent tool for multiple gene expression, for studies of their functions, and their applications in insect species.

Drug-Conjugated Dendrimer Hydrogel Enables Sustained Drug Release via a Self-Cleaving Mechanism.

In this study, the anticancer drug, camptothecin (CPT), was covalently grafted onto polyamidoamine (PAMAM) dendrimer surface and then reacted with polyethylene glycol diacrylate (PEG-DA) to form dendrimer hydrogel (DH-G3-CPT) with low cross-linking density. In this novel drug delivery system, CPT was cleaved from dendrimer via the ammonolysis of ester bonds and then diffused out of the hydrogel network, thus leading to significantly prolonged drug release. The self-cleaving release kinetics of camptothecin can be further tuned by pH. This DH-G3-CPT drug delivery system has both injectability and sustained drug release. It showed an excellent tumor inhibition effect following intratumoral injection in a head and neck cancer model of mouse.

A Peptide-Induced Self-Cleavage Reaction Initiates the Activation of Tyrosinase.

The conversion of inactive pro-polyphenol oxidases (pro-PPOs) into the active enzyme results from the proteolytic cleavage of its C-terminal domain. Herein, a peptide-mediated cleavage process that activates pro-MdPPO1 (Malus domestica) is reported. Mass spectrometry, mutagenesis studies, and X-ray crystal-structure analysis of pro-MdPPO1 (1.35 Å) and two separated C-terminal domains, one obtained upon self-cleavage of pro-MdPPO1 and the other one produced independently, were applied to study the observed self-cleavage. The sequence Lys 355-Val 370 located in the linker between the active and the C-terminal domain is indispensable for the self-cleavage. Partial introduction (Lys 352-Ala 360) of this peptide into the sequence of two other PPOs, MdPPO2 and aurone synthase (CgAUS1), triggered self-cleavage in the resulting mutants. This is the first experimental proof of a self-cleavage-inducing peptide in PPOs, unveiling a new mode of activation for this enzyme class that is independent of any external protease.

Biology of Trypanosoma cruzi Retrotransposons: From an Enzymatic to a Structural Point of View.

INTRODUCTION: An important portion of the Trypanosoma cruzi genome is composed of mobile genetic elements, which are interspersed with genes on all chromosomes. The L1Tc non-LTR retrotransposon and its truncated version NARTc are the most highly represented and best studied of these elements. L1Tc is actively transcribed in all three forms of the Trypanosoma parasite and encodes the proteins that enable it to autonomously mobilize. This mini review discusses the enzymatic properties of L1Tc that enable its mobilization and possibly the mobilization of other non-autonomous retrotransposons in Trypanosoma. We also briefly review the Hepatitis Delta Virus-like autocatalytic and 2A self-cleaving viral-like sequences contained in L1Tc that regulate post-transcriptional properties such as relative protein abundance and mRNA stability. Special emphasis is placed on the Pr77 dual system, which is based on the RNA pol II-dependent internal promoter of L1Tc and NARTc and the HDV-like ribozyme activity encoded by the first 77 nucleotides of the element's DNA and RNA. The high degree of conservation of the Pr77 sequence, referred to as the "Pr77-hallmark", among different trypanosomatid retroelements suggests that these mobile elements are responsible for the distribution of regulatory sequences within the genome they inhabit. CONCLUSION: We also discuss how the involvement of L1Tc and NARTc in the gene regulatory processes of these parasites could justify their domestication and long-term coexistence in these ancient organisms.

Identification of 15 candidate structured noncoding RNA motifs in fungi by comparative genomics.

BACKGROUND: With the development of rapid and inexpensive DNA sequencing, the genome sequences of more than 100 fungal species have been made available. This dataset provides an excellent resource for comparative genomics analyses, which can be used to discover genetic elements, including noncoding RNAs (ncRNAs). Bioinformatics tools similar to those used to uncover novel ncRNAs in bacteria, likewise, should be useful for searching fungal genomic sequences, and the relative ease of genetic experiments with some model fungal species could facilitate experimental validation studies. RESULTS: We have adapted a bioinformatics pipeline for discovering bacterial ncRNAs to systematically analyze many fungal genomes. This comparative genomics pipeline integrates information on conserved RNA sequence and structural features with alternative splicing information to reveal fungal RNA motifs that are candidate regulatory domains, or that might have other possible functions. A total of 15 prominent classes of structured ncRNA candidates were identified, including variant HDV self-cleaving ribozyme representatives, atypical snoRNA candidates, and possible structured antisense RNA motifs. Candidate regulatory motifs were also found associated with genes for ribosomal proteins, S-adenosylmethionine decarboxylase (SDC), amidase, and HexA protein involved in Woronin body formation. We experimentally confirm that the variant HDV ribozymes undergo rapid self-cleavage, and we demonstrate that the SDC RNA motif reduces the expression of SAM decarboxylase by translational repression. Furthermore, we provide evidence that several other motifs discovered in this study are likely to be functional ncRNA elements. CONCLUSIONS: Systematic screening of fungal genomes using a computational discovery pipeline has revealed the existence of a variety of novel structured ncRNAs. Genome contexts and similarities to known ncRNA motifs provide strong evidence for the biological and biochemical functions of some newly found ncRNA motifs. Although initial examinations of several motifs provide evidence for their likely functions, other motifs will require more in-depth analysis to reveal their functions.

High-throughput purification of recombinant proteins using self-cleaving intein tags.

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (ßGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.

Numerous small hammerhead ribozyme variants associated with Penelope-like retrotransposons cleave RNA as dimers.

Hammerhead ribozymes represent the most common of the 9 natural classes of self-cleaving RNAs. The hammerhead catalytic core includes 11 highly-conserved nucleotides located largely within the unpaired regions of a junction formed by stems I, II and III. The vast majority of previously reported examples carry an additional pseudoknot or other tertiary interactions between nucleotides that precede stem I and nucleotides in the loop of stem II. These extra contacts are critical for high-speed RNA catalysis. Herein, we report the discovery of ∼150,000 additional variant hammerhead representatives that exhibit diminished stem III substructures. These variants are frequently associated with Penelope-like retrotransposons, which are a type of mobile genetic element. Kinetic analyses indicate that these RNAs form dimers to cleave RNA.

The Hammerhead Ribozyme: A Long History for a Short RNA.

Small nucleolytic ribozymes are a family of naturally occurring RNA motifs that catalyse a self-transesterification reaction in a highly sequence-specific manner. The hammerhead ribozyme was the first reported and the most extensively studied member of this family. However, and despite intense biochemical and structural research for three decades since its discovery, the history of this model ribozyme seems to be far from finished. The hammerhead ribozyme has been regarded as a biological oddity typical of small circular RNA pathogens of plants. More recently, numerous and new variations of this ribozyme have been found to inhabit the genomes of organisms from all life kingdoms, although their precise biological functions are not yet well understood.

Intein applications: from protein purification and labeling to metabolic control methods.

The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification.

Gene Stacking and Stoichiometric Expression of ER-Targeted Constructs Using "2A" Self-Cleaving Peptides.

Simultaneous stoichiometric expression of multiple genes plays a major part in modern research and biotechnology. Traditional methods for incorporating multiple transgenes (or "gene stacking") have drawbacks such as long time frames, uneven gene expression, gene silencing, and segregation derived from the use of multiple promoters. 2A self-cleaving peptides have emerged over the last two decades as a functional gene stacking method and have been used in plants for the co-expression of multiple genes under a single promoter. Here we describe design features of multicistronic polyproteins using 2A peptides for co-expression in plant cells and targeting to the endoplasmic reticulum (ER). We designed up to quad-cistronic vectors that could target proteins in tandem to the ER. We also exemplify the incorporation of self-excising intein domains within 2A polypeptides, to remove residue additions. These features could aid in the design of stoichiometric protein co-expression strategies in plants in combination with targeting to different subcellular compartments.

A compact, versatile drug-induced splicing switch system with minimal background expression.

Gene-switch techniques hold promising applications in contemporary genetics research, particularly in disease treatment and genetic engineering. Here, we developed a compact drug-induced splicing system that maintains low background using a human ubiquitin C (hUBC) promoter and optimized drug (LMI070) binding sequences based on the Xon switch system. To ensure precise subcellular localization of the protein of interest (POI), we inserted a 2A self-cleaving peptide between the extra N-terminal peptide and POI. This streamlined and optimized switch system, named miniXon2G, effectively regulated POIs in different subcellular localizations both in vitro and in vivo. Furthermore, miniXon2G could be integrated into endogenous gene loci, resulting in precise, reversible regulation of target genes by both endogenous regulators and drugs. Overall, these findings highlight the performance of miniXon2G in controlling protein expression with great potential for general applicability to diverse biological scenarios requiring precise and delicate regulation.

CRISPR-Cas9-Mediated Bioluminescent Tagging of Endogenous Proteins by Fluorescent Protein-Assisted Cell Sorting.

Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC). HiBiT is an 11-amino acid peptide derived from the NanoLuc luciferase that produces a luminescence signal which is ~100-fold brighter than firefly or Renilla luciferases through high-affinity binding to a complementary NanoLuc peptide fragment called LgBiT. To facilitate single-cell clonal isolation of knock-ins, the homology-directed repair template encoding HiBiT was followed by a P2A self-cleaving peptide for coexpression of an mCherry fluorescent protein as a fluorescence-activated cell sorter (FACS)-selectable marker. HiBiT tagging thus allows highly sensitive luminescence detection of endogenous PAX3-FOXO1 levels permitting quantitative high-throughput screening of large compound libraries for the discovery of PAX3-FOXO1 inhibitors and degraders.

Ligation-independent cloning and self-cleaving intein as a tool for high-throughput protein purification.

The rapid production of purified recombinant proteins has become increasingly important for countless applications. Many purification methods involve expression of target proteins in fusion to purification tags, which often must be removed from the target proteins after purification. Recently, engineered inteins have been used to create convenient self-cleaving tags for tag removal. Although intein methods can greatly simplify protein purification, commercially available expression vectors still rely on conventional restriction/ligation cloning methods for target gene insertion. We have streamlined this process by introducing Ligation-Independent Cloning (LIC) capability to our intein expression plasmids, which provides a simple method for constructing self-cleaving tag-target gene fusions. In this work, we demonstrate efficient gene insertion via this system, as well as target protein expression and purification consistent with previously reported results. Through this newly developed system, arbitrary protein genes can be rapidly incorporated into self-cleaving tag expression vectors, and their products purified using convenient platform methods.