Foreign DNA Fragments Research Articles

NHEJ inhibitor SCR7 and its different forms: Promising CRISPR tools for genome engineering.

The CRISPR-Cas system currently stands as one of the best multifaceted tools for site-specific genome engineering in mammals. An important aspect of research in this field focusses on improving the specificity and efficacy of precise genome editing in multiple model systems. The cornerstone of this mini-review is one of the extensively investigated small molecule inhibitor, SCR7, which abrogates NHEJ, a Ligase IV-dependent DSB repair pathway, thus guiding integration of the foreign DNA fragment via the more precise homology directed repair during genome editing. One of our recent studies sheds light on properties of different forms of SCR7. Here, we give a succinct account on the use of SCR7 and its different forms in CRISPR-Cas system, highlighting their chemical properties and biological relevance as potent efficiency-enhancing CRISPR tools.

Protocol for Agrobacterium-mediated transformation of tall fescue and future perspective on the application of genome editing.

Tall fescue (Festuca arundinacea Schreb.) is a major cool-season perennial grass grown for forage and turf. We have obtained transgenic tall fescue by Agrobacterium-mediated transformation to improve agronomically important traits. In our protocol, we use embryogenic calli derived from not only mature seeds but also shoot tips. Although tall fescue cultivars consist of various genotypes with different genetic variation, we can produce transgenic plants at any time with calli induced from shoot tips of in vitro-maintained responsive genotypes. When the hygromycin phosphotransferase gene is used as a selectable marker, transformants are selected by incubation with 100 mg l-1 hygromycin in both selection and regeneration media. Since tall fescue is an anemophilous species, the cultivation of transgenic plants poses the risk of transgenic pollen flow. Recently, it has been reported that genome-edited plants without the integration of foreign DNA fragments can be produced by an Agrobacterium-mediated transient gene expression system. We hope that our protocol will contribute to production of transgene-free genome-edited tall fescue.

Generation of Transgenic Rats using a Lentiviral Vector Approach.

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3' overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3'-5' exonucleases DnaQ and ExoT can trim long 3' overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3'-5' exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.

Selective loading and processing of prespacers for precise CRISPR adaptation

CRISPR-Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

Possible existence of a system similar to bacterial restriction–modification in plants

The bacterial restriction–modification (R-M) system has evolved as a defense mechanism against infectious phages and other types of integration of foreign DNA fragments into the host chromosome through recombination. In this study, we identified two endonucleases (wheat endonuclease 1-WEN1 and wheat endonuclease 2-WEN2) and the adenine-DNA methyltransferase WAD (involved in mitochondrial DNA methylation) in the vesicular fraction of coleoptiles of wheat Triticum aestivum. WEN1 and WEN2 have multidirectional sensitivity to DNA methylation and to the presence of S-adenosyl-L-methionine and Ca2+ ions, which suggests their participation in the protection of mitochondrial DNA, similar to bacterial DNA. WEN2 has a GAT recognition site, which is part of the methylation site of adenine DNA methyltransferase (WAD-TGATCA) and hydrolyzes the CG (GC) bond upstream of the recognition site only if it is not methylated. Based on the interrelations between the two enzymes, we propose that an R-M system similar to that of the type IV observed in bacteria may exist in plants.

Screening Bacterial Colonies Using X-Gal and IPTG: α-Complementation.

Many plasmid vectors (e.g., the pUC series, Bluescript, pGem, and their derivatives) carry a short segment of Escherichia coli DNA containing the regulatory sequences and the coding information for the first 146 amino acids of β-galactosidase. Vectors of this type are used in host cells that express the carboxy-terminal portion of β-galactosidase. Although neither the host-encoded fragments nor the plasmid-encoded fragments of β-galactosidase are themselves active, they can associate to form an enzymatically active protein. This type of complementation, in which deletion mutants of the operator-proximal segment of the lacZ gene are complemented by β-galactosidase-negative mutants that have the operator-proximal region intact, is called α-complementation. The lac+ bacteria that result from α-complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-Gal. However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an amino-terminal fragment that is no longer capable of α-complementation. Bacteria carrying recombinant plasmids therefore form white colonies. To screen bacterial colonies, the chromogenic substrate X-Gal and the gratuitous inducer IPTG are mixed with suitable dilution of a culture, combined with molten top agar, and then spread on agar plates containing the appropriate antibiotic. The efficiency of transformation is slightly higher when the bacteria are plated in top agar rather than on the surface of agar plates. Perhaps the transformed bacteria prefer the slightly anaerobic state within the soft agar or the isosmolarity provided by the agar medium.

Current status on the modification of the scope for GMO regulation on the gene edited plants with no remnants of inserted foreign DNA fragments

Current status on the modification of the scope for GMO regulation on the gene edited plants with no remnants of inserted foreign DNA fragments

A Functional Mini-Integrase in a Two-Protein Type V-C CRISPR System

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrasecomprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.

Biolistic Delivery of CRISPR/Cas9 with Ribonucleoprotein Complex in Wheat.

The great advances in exploiting the CRISPR/Cas9 system are paving the way for targeted genome engineering in plants. Genome editing by direct delivery of CRISPR/Cas9 ribonucleoprotein complexes (RNPs) into plant cells reduces off-target mutations and avoids the integration of foreign DNA fragments, thus providing an efficient and accurate method for precision crop breeding. Here we describe an RNP-based genome editing protocol for wheat. The protocol covers the in vitro transcription of sgRNA, purification of Cas9 protein, biolistic delivery of CRISPR/Cas9 RNPs, and tissue culture procedures for regenerating testable seedlings.

Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation

CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide-a consensus PAM-within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations.IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a "directionality" in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.

Molecular mechanisms of CRISPR–Cas spacer acquisition

Many bacteria and archaea have the unique ability to heritably alter their genomes by incorporating small fragments of foreign DNA, called spacers, into CRISPR loci. Once transcribed and processed into individual CRISPR RNAs, spacer sequences guide Cas effector nucleases to destroy complementary, invading nucleic acids. Collectively, these two processes are known as the CRISPR-Cas immune response. In this Progress article, we review recent studies that have advanced our understanding of the molecular mechanisms underlying spacer acquisition and that have revealed a fundamental link between the two phases of CRISPR immunity that ensures optimal immunity from newly acquired spacers. Finally, we highlight important open questions and discuss the potential basic and applied impact of spacer acquisition research.

DnaQ exonuclease-like domain of Cas2 promotes spacer integration in a type I-E CRISPR-Cas system.

CRISPR-Cas systems constitute an adaptive immune system that provides acquired resistance against phages and plasmids in prokaryotes. Upon invasion of foreign nucleic acids, some cells integrate short fragments of foreign DNA as spacers into the CRISPR locus to memorize the invaders and acquire resistance in the subsequent round of infection. This immunization step called adaptation is the least understood part of the CRISPR-Cas immunity. We have focused here on the adaptation stage of Streptococcus thermophilus DGCC7710 type I-E CRISPR4-Cas (St4) system. Cas1 and Cas2 proteins conserved in nearly all CRISPR-Cas systems are required for spacer acquisition. The St4 CRISPR-Cas system is unique because the Cas2 protein is fused to an additional DnaQ exonuclease domain. Here, we demonstrate that St4 Cas1 and Cas2-DnaQ form a multimeric complex, which is capable of integrating DNA duplexes with 3'-overhangs (protospacers) in vitro We further show that the DnaQ domain of Cas2 functions as a 3'-5'-exonuclease that processes 3'-overhangs of the protospacer to promote integration.

Solution structure and dynamics of Xanthomonas albilineans Cas2 provide mechanistic insight on nuclease activity.

Cas2 protein in the CRISPR-Cas system functions as a scaffold for the acquisition of foreign DNA fragments, and as a nuclease against DNA and RNA substrates. Crystal structures of Cas2 have shown catalytically inactive conformational states that do not explain the mechanism of Cas2 nuclease activity. Here, we report that Xanthomonas albilineans Cas2 (XaCas2) assumes an inactive conformation in solution. Residual dipolar couplings and NMR relaxation, however, provide direct evidence on conformational dynamics at the predicted hinge region. Furthermore, XaCas2 transiently associates with metal ions for nuclease activity via highly mobile Asp8. Taken together, the dual function of Cas2 can be explained by a dynamic equilibrium of conformational states that serve as a scaffold or as a nuclease on demand.

Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus.

To acquire CRISPR–Cas immunity against invasive mobile genetic elements, prokaryotes must first integrate fragments of foreign DNA into their genomic CRISPR arrays for use in future invader silencing. Here, we found that the hyperthermophilic archaeaon, Pyrococcus furiosus, actively incorporates DNA fragments (spacers) from both plasmid (foreign) and host genome (self) sequences into its seven CRISPR loci. The majority of new spacers were derived from DNA immediately downstream from a 5′-CCN-3′ protospacer adjacent motif (PAM) that is critical for invader targeting. Interestingly, spacers were preferentially acquired from genome or plasmid regions corresponding to active transposons, CRISPR loci, ribosomal RNA genes, rolling circle origins of replication, and areas where plasmids recombined with the host chromosome. A common feature of the highly sampled spacers is that they arise from DNA regions expected to undergo DNA nicking and/or double-strand breaks. Taken together with recent results from bacterial systems, our findings indicate that free DNA termini and PAMs are conserved features important for CRISPR spacer uptake in diverse prokaryotes and CRISPR–Cas systems. Moreover, lethal self-targeting by CRISPR systems may contribute to host genome stability by eliminating cells undergoing active transposon mobility or chromosomal uptake of autonomously replicating foreign mobile genetic elements.

Genetic determinant of Bacillus pumilus lipase lethality and its application as positive selection cloning vector in Escherichia coli

Positive selection vectors carry genes that upon expression produce proteins that cause host cell deaths. Insertion of foreign DNA fragments within the ORF of the gene disrupts the lethal effect of the expressed protein. This study described the cloning of Family I.4 Bacillus pumilus lipase gene whose expressed protein is toxic and lethal to Escherichia coli JM109 (DE3) cells. The determinant of toxicity was identified through Error-prone PCR to be the nature of amino acid residue resident at position 28 of the mature lipase protein. The presence of Thr/Ser28 within the mature lipases of B. pumilus and B. licheniformis resulted in lethality to E. coli cells. However, the Thr28Ala or Thr28Gly mutations relieved the lethal phenotype of mature Family I.4 Bacillus lipases. The toxic effect of the expressed mature B. pumilus lipase protein was exploited in the development of a positive selection cloning vector. The B. pumilus lipase gene was synthesised to contain 13 unique silent restriction sites within the ORF, and placed under the regulation of T7 promoter of the pET expression system. Insertional inactivation of the gene's toxic protein was achieved by cloning DNA fragments of different sizes within the designed multiple cloning sites. The toxic effect of the lipase protein was disrupted indicating the potential of the gene for application in suicidal positive selection cloning vectors. The results revealed that protein expression and engineering studies aimed at optimal production of mature Family I.4 Bacillus lipases in E. coli should take into consideration the nature of amino acid 28 resident.

Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in Escherichia coli.

To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose. The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the PR promoter and kanamycin resistance gene (PR-kan R ) at 30°C, but have no effect on PR-kan R gene at 37°C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~107 cfu per µg of genomic DNA, with no empty vectors detected. Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.

Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation

During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR–Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1–Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.

PELMO, an optimised in-house cloning vector

DNA cloning is an essential tool regarding DNA recombinant technology as it allows the replication of foreign DNA fragments within a cell. pELMO was here constructed as an in-house cloning vector for rapid and low-cost PCR product propagation; it is an optimally designed vector containing the ccdB killer gene from the pDONR 221 plasmid, cloned into the pUC18 vector’s multiple cloning site (Thermo Scientific). The ccdB killer gene has a cleavage site (CCC/GGG) for the SmaI restriction enzyme which is used for vector linearisation and cloning blunt-ended products. pELMO transformation efficiency was evaluated with different sized inserts and its cloning efficiency was compared to that of the pGEM-T Easy commercial vector. The highest pELMO transformation efficiency was observed for ~500 bp DNA fragments; pELMO vector had higher cloning efficiency for all insert sizes tested. In-house and commercial vector cloned insert reads after sequencing were similar thus highlighting that sequencing primers were designed and localised appropriately. pELMO is thus proposed as a practical alternative for in-house cloning of PCR products in molecular biology laboratories.

P.1.a.014 - MicroRNA expression after a single treatment with an anxiogenic dose of pilocarpine in rats

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T0 plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.