Foreign DNA Fragments Research Articles

Systematic comparison of genome information processing and boundary recognition tools used for genomic island detection

Genomic islands are fragments of foreign DNA that are found in bacterial and archaeal genomes, and are typically associated with symbiosis or pathogenesis. While numerous genomic island detection methods have been proposed, there has been limited evaluation of the efficiency of the genome information processing and boundary recognition tools. In this study, we conducted a review of the statistical methods involved in genomic signatures, host signature extraction, informative signature selection, divergence measures, and boundary detection steps in genomic island prediction. We compared the performances of these methods on simulated experiments using alien fragments obtained from both artificial and real genomes. Our results indicate that among the nine genomic signatures evaluated, genomic signature frequency and full probability performed the best. However, their performance declined when normalized to their expectations and variances, such as Z-score and composition vector. Based on our experiments of the E. coli genome, we found that the confidence intervals of the window variances achieved the best performance in the signature extraction of the host, with the best confidence interval being 1.5–2 times the standard error. Ordered kurtosis was most effective in selecting informative signatures from a single genome, without requiring prior knowledge from other datasets. Among the three divergence measures evaluated, the two-sample t-test was the most successful, and a non-overlapping window with a small eye window (size 2) was best suited for identifying compositionally distinct regions. Finally, the maximum of the Markovian Jensen-Shannon divergence score, in terms of GC-content bias, was found to make boundary detection faster while maintaining a similar error rate.

Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays.

Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560-kDa integration complex structure that explains how Pseudomonas aeruginosa Cas (Cas1-Cas2/3) and non-Cas proteins (for example, integration host factor) fold 150 base pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1-2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1-2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3 proteins rotate 100 degrees to expose DNA-binding sites on either side of the Cas2 homodimer, which each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1-2/3-mediated integration to diverse repeat sequences that have a 5'-GT. Collectively, this work reveals new DNA-binding surfaces on Cas2 that are critical for DNA folding and site-specific delivery of foreign DNA.

Establishment of a transformation system in close relatives of wheat under the assistance of TaWOX5

Species closely related to wheat are important genetic resources for agricultural production, functional genomics studies and wheat improvement. In this study, a wheat gene related to regeneration, TaWOX5, was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum, hexaploid triticale, and rye (Secale cereale L.) using their immature embryos. Transgenic plants were efficiently generated. During the transformation process, the Agrobacterium infection efficiency was assessed by histochemical staining for β-glucuronidase (GUS). Finally, the transgenic nature of regenerated plants was verified by polymerase chain reaction (PCR)-based genotyping for the presence of the GUS and bialaphos resistance (bar) genes, histochemical staining for GUS protein, and the QuickStix strip assay for bar protein. The transformation efficiency of T. monococcum genotype PI428182 was 94.4%; the efficiencies of four hexaploid triticale genotypes Lin456, ZS3297, ZS1257, and ZS3224 were 52.1, 41.2, 19.4, and 16.0%, respectively; and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) analyses indicated that the GUS transgenes were integrated into the distal or near centromere (proximal) regions of the chromosomes in transgenic T. monococcum and hexaploid triticale plants. In the transgenic hexaploid triticale plants, the foreign DNA fragment was randomly integrated into the AABB and RR genomes. Furthermore, the transgene was almost stably inherited in the next generation by Mendel’s law. The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.

Basic Characterization of Natural Transformation in Avibacterium paragallinarum.

Avibacterium paragallinarum is the pathogen involved in infectious coryza (IC), an acute infectious upper respiratory disease in chickens. The prevalence of IC has increased in China in recent years. There is a lack of reliable and effective procedures for gene manipulation, which has limited the research on the bacterial genetics and pathogenesis of A. paragallinarum. Natural transformation has been developed as a method of gene manipulation in Pasteurellaceae by the introduction of foreign genes or DNA fragments into bacterial cells, but there has been no report on natural transformation in A. paragallinarum. In this study, we analyzed the existence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum and established a method for transformation in it. Through bioinformatics analysis, we identified 16 homologs of Haemophilus influenzae competence proteins in A. paragallinarum. We found that the uptake signal sequence (USS) was overrepresented in the genome of A. paragallinarum (1,537 to 1,641 copies of the core sequence ACCGCACTT). We then constructed a plasmid, pEA-KU, that carries the USS and a plasmid, pEA-K, without the USS. These plasmids can be transferred via natural transformation into naturally competent strains of A. paragallinarum. Significantly, the plasmid that carries USS showed a higher transformation efficiency. In summary, our results demonstrate that A. paragallinarum has the ability to undergo natural transformation. These findings should prove to be a valuable tool for gene manipulation in A. paragallinarum. IMPORTANCE Natural transformation is an important mechanism for bacteria to acquire exogenous DNA molecules during the process of evolution. Additionally, it can also be used as a method to introduce foreign genes into bacteria under laboratory conditions. Natural transformation does not require equipment such as an electroporation apparatus. It is easy to perform and is similar to gene transfer under natural conditions. However, there have been no reports on natural transformation in Avibacterium paragallinarum. In this study, we analyzed the presence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum. Our results indicate that natural competence could be induced in A. paragallinarum serovars A, B, and C. Furthermore, the method that we established to transform plasmids into naturally competent A. paragallinarum strains was stable and efficient.

Development of a set of novel binary expression vectors for plant gene function analysis and genetic transformation.

With the advent of multiple omics and Genome-Wide Association Studies (GWAS) technology, genome-scale functional analysis of candidate genes is to be conducted in diverse plant species. Construction of plant binary expression vectors is the prerequisite for gene function analysis. Therefore, it is of significance to develop a set of plant binary expression vectors with highly efficient, inexpensive, and convenient cloning method, and easy-to-use in screening of positive recombinant in Escherichia coli. In this study, we developed a set of plant binary expression vectors, termed pBTR vectors, based on Golden Gate cloning using BsaI restriction site. Foreign DNA fragment of interest (FDI) can be cloned into the destination pBTR by one-step digestion-ligation reaction in a single tube, and even the FDI contains internal BsaI site(s). Markedly, in one digestion-ligation reaction, multiple FDIs (exemplified by cloning four soybean Glyma.02g025400, Glyma.05g201700, Glyma.06g165700, and Glyma.17g095000 genes) can be cloned into the pBTR vector to generate multiple corresponding expression constructs (each expression vector carrying an FDI). In addition, the pBTR vectors carry the visual marker, a brightness monomeric red fluorescent protein mScarlet-I, that can be observed with the unaided eye in screening of positive recombinants without the use of additional reagents/equipment. The reliability of the pBTR vectors was validated in plants by overexpression of AtMyb75/PAP1 in tomato and GUSPlus in soybean roots via Agrobacterium rhizogenes-mediated transformation, promoter activity analysis of AtGCSpro in Arabidopsis via A. tumefaciens-mediated transformation, and protein subcellular localization of the Vitis vinifera VvCEB1opt in tobacco, respectively. These results demonstrated that the pBTR vectors can be used in analysis of gene (over)expression, promoter activity, and protein subcellular localization. These vectors will contribute to speeding up gene function analysis and the process of plant molecular breeding.

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.

Genome editing toolboxes are essential for the exploration and exploitation of non-conventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The non-conventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1,000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses non-homologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.

Characterization of a novel Escherichia coli recombineering selection/counterselection cassette

Recombineering is a highly efficient DNA cloning and modification technique by using the recombinase-mediated homologous recombination. Selection/counterselection cassette is often used in chromosomal DNA or large episomal DNA manipulation, in which the selection marker is used for the first step cassette selection while deleting the target gene via allelic exchange, and the counterselection marker is used for the second step replacement of the cassette by the foreign DNA fragment. A variety of selection/counterselection cassettes are reported, however, the cassettes suffer from the shortcomings of the requirement of pre-engineered strain or specific culture medium. Herein, we report a novel S-tetR- PtetA-ccdB-aacC1-S selection/counterselection cassette that sidesteps the disadvantages. As a proof-of-concept, one-step gene cloning (0.7, 1.7, and 4.2kb) and two-step Escherichia coli chromosomal gene knock-in (0.7 and 4.2kb) were performed. The gene cloning and gene knock-in efficiencies are high up to 90%. The novel selection/counterselection cassette adds a powerful tool to the recombineering repertoire.

A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/Cas14 Proteins Utilized for Genome Engineering.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system has altered life science research offering enormous options in manipulating, detecting, imaging, and annotating specific DNA or RNA sequences of diverse organisms. This system incorporates fragments of foreign DNA (spacers) into CRISPR cassettes, which are further transcribed into the CRISPR arrays and then processed to make guide RNA (gRNA). The CRISPR arrays are genes that encode Cas proteins. Cas proteins provide the enzymatic machinery required for acquiring new spacers targeting invading elements. Due to programmable sequence specificity, numerous Cas proteins such as Cas9, Cas12, Cas13, and Cas14 have been exploited to develop new tools for genome engineering. Cas variants stimulated genetic research and propelled the CRISPR/Cas tool for manipulating and editing nucleic acid sequences of living cells of diverse organisms. This review aims to provide detail on two classes (class 1 and 2) of the CRISPR/Cas system, and the mechanisms of all Cas proteins, including Cas12, Cas13, and Cas14 discovered so far. In addition, we also discuss the pros and cons and recent applications of various Cas proteins in diverse fields, including those used to detect viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This review enables the researcher to gain knowledge on various Cas proteins and their applications, which have the potential to be used in next-generation precise genome engineering.

Similarities of P1-Like Phage Plasmids and Their Role in the Dissemination of blaCTX-M-55.

ABSTRACTThe P1-like phage plasmid (PP) has been widely used as a molecular biology tool, but its role as an active accessory cargo element is not fully understood. In this study, we provide insights into the structural features and gene content similarities of 77 P1-like PPs in the RefSeq database. We also describe a P1-like PP carrying a blaCTX-M-55 gene, JL22, which was isolated from a clinical strain of Escherichia coli from a duck farm. P1-like PPs were very similar and conserved based on gene content similarities, with only eight highly variable regions. Importantly, two kinds of replicon types, namely, IncY and p0111, were identified and can be used to specifically identify the P1-like phage. JL22 is similar to P1, acquiring an important foreign DNA fragment with two obvious features, namely, the plasmid replication gene repA′ (p0111) replacing the gene repA (IncY) and a 4,200-bp fragment mobilized by IS1380 and IS5 and containing a blaCTX-M-55 gene and a trpB gene encoding tryptophan synthase (indole salvaging). The JL22 phage could be induced but had no lytic capacities. However, a lysogenic recipient and intact structure of JL22 virions were observed, showing that the extended-spectrum β-lactamase blaCTX-M-55 gene was successfully transferred. Overall, conserved genes can be a good complement to improve the identification efficiency and accuracy in future screening for P1-like PPs. Moreover, the highly conserved structures may be important for their prevalence and dissemination.IMPORTANCE As a PP, P1 DNA exists as a low-copy-number plasmid and replicates autonomously with a lysogenization style. This unique mode of P1-like elements probably indicates a stable contribution to antibiotic resistance. After analyzing these elements, we show that P1-like PPs are very similar and conserved, with only eight highly variable regions. Moreover, we observed the occurrence of replicon IncY and p0111 only in the P1-like PP community, implying that these conserved regions, coupled with IncY and p0111, can be an important complement in future screening of P1-like PPs. Identification and characterization of JL22 confirmed our findings that major changes were located in variable regions, including the first detection of blaCTX-M-55 in such a mobile genetic element. This suggests that these variable regions may facilitate foreign DNA mobilization. This study features a comprehensive genetic analysis of P1-like PPs, providing new insights into the dissemination mechanisms of antibiotic resistance through P1 PPs.

CTREP-finder: A web service for quick identification and visualization of clean transgenic and genome-edited plants

To feed 10 billion people in 30 years from now will heavily depend on new higher yield, yet safe, biotech crop varieties, which are developed using biotechnologies, particularly genetic transformation and genome editing. A clean transgenic plant (CTP) contains only the defined transgene, whilst a clean edited plant (CEP) is transgene-free. Currently, it is difficult and time-consuming to identify and locate transgenes within plant genomes due to the complex nature of transfer DNA (T-DNA) or plasmid backbone integration, which has led to a major obstacle in mass scale production of clean transgenic/edited plants (CTREPs). Here we have built a web-based portal, CTREP-finder, for fast detection, characterization and visualization of foreign DNA fragments in plants, based on next-generation whole genome sequencing data. We invented a novel bioinformatics strategy to handle three scenarios of transgene integration and successfully identified CTREPs by testing 132 samples. Furthermore, we compared the CTREP-finder with three public programs, FED, transgeneR and TDNAscan, and it showed that the CTREP-finder outperforms those programs particularly on the localization of transgene integration. The use of CTREP-finder needs little requirement for bioinformatics expertise. Therefore, it is a user-friendly web-based portal that could be used by plant breeders and regulatory agencies for the rapid selection/detection of CTREPs for crop breeding and production.

Fungal insecticidal activity elevated by non-risky markerless overexpression of an endogenous cysteine-free protein gene in Beauveria bassiana.

Fungal insecticides are notorious for slow kill action, an intrinsic trait that can be improved by the genetic engineering of an exogenous or endogenous virulence factor. However, transgenic insecticides expressing exogenous toxin and herbicide-resistant marker genes may cause unexpected ecological risks and are hardly permitted for field release due to strict regulatory hurdles. It is necessary to improve biotechnology that can speed up fungal insect-killing action and exclude ecological risk source. A markerless transformation system of Beauveria bassiana, a main source of wide-spectrum fungal insecticides, was reconstructed based on the fungal uridine auxotrophy (Δura3). The system was applied for overexpression of the small cysteine-free protein (120 amino acids) gene cfp previously characterized as a regulator of the fungal virulence and gene expression. Three cfp-overexpressed strains showed much faster kill action to Galleria mellonella larvae than the parental wild-type via normal cuticle infection but no change in vegetative growth and aerial condition. The faster kill action was achieved due to not only significant increases in conidial adherence to insect cuticle and total activity of secreted cuticle-degrading Pr1 proteases and of antioxidant enzymes crucial for collapse of insect immune defense but acceleration of hemocoel localization, proliferation in vivo and host death from mummification. The markerless system is free of any foreign DNA fragment as a source of ecologic risk and provides a novel biotechnological approach to enhancing fungal insecticidal activity with non-risky endogenous genes and striding over the regulatory hurdles. © 2022 Society of Chemical Industry.

Photo-Cross-Linked Probe-Modified Magnetic Particles for the Selective and Reliable Recovery of Nucleic Acids.

Polymerase chain reaction (PCR) assays are used to diagnose various infectious diseases such as Coronavirus disease 2019 by detecting the nucleic acids of the pathogen. However, in practice, the yield of the extraction process and the inhibition of the reverse transcription reaction and PCR by foreign substances reduce the sensitivity and may yield false negative results. The sensitivity of the PCR test can be improved by using technologies that can reliably capture the target nucleic acid and remove foreign substances. In this study, we developed photo-cross-linkable probe-modified magnetic particles (PPMPs) for the sequence-specific recovery of target nucleic acids using photo-cross-linkable artificial nucleic acid probes and magnetic particles. Nucleic acid probes modified with photo-cross-linkable artificial nucleic acids can hybridize with the target nucleic acids in a sequence-specific manner and then securely capture the target nucleic acids by UV irradiation-mediated covalent bonding. Then the target nucleic acid is detected by trapping the target-bound probe on the surface of the magnetic particles and subjecting these collected magnetic particles to PCR. Recovery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene pseudo-DNA (120 bp) was performed using PPMPs. We confirmed that the PPMPs captured the target consistently even after washes were done with denaturing agents and surfactants. Even in the presence of foreign DNA fragments, PPMPs were able to specifically recover the target DNA. This method allows for a more accurate detection by recovering only the target DNA for PCR. Hence, PPMPs can be successfully used for PCR-mediated detection of SARS-CoV-2 and other pathogens whose nucleic acid sequences are known.

Whole-Genome Transformation of Yeast Promotes Rare Host Mutations with a Single Causative SNP Enhancing Acetic Acid Tolerance.

ABSTRACTWhole-genome (WG) transformation (WGT) with DNA from the same or another species has been used to obtain strains with superior traits. Very few examples have been reported in eukaryotes—most apparently involving integration of large fragments of foreign DNA into the host genome. We show that WGT of a haploid acetic acid-sensitive Saccharomyces cerevisiae strain with DNA from a tolerant strain, but not from nontolerant strains, generated many tolerant transformants, some of which were stable upon subculturing under nonselective conditions. The most tolerant stable transformant contained no foreign DNA but only seven nonsynonymous single nucleotide polymorphisms (SNPs), of which none was present in the donor genome. The SNF4 mutation c.[805G→T], generating Snf4E269*, was the main causative SNP. Allele exchange of SNF4E269* or snf4Δ in industrial strains with unrelated genetic backgrounds enhanced acetic acid tolerance during fermentation under industrially relevant conditions. Our work reveals a surprisingly small number of mutations introduced by WGT, which do not bear any sequence relatedness to the genomic DNA (gDNA) of the donor organism, including the causative mutation. Spontaneous mutagenesis under protection of a transient donor gDNA fragment, maintained as extrachromosomal circular DNA (eccDNA), might provide an explanation. Support for this mechanism was obtained by transformation with genomic DNA of a yeast strain containing NatMX and selection on medium with nourseothricin. Seven transformants were obtained that gradually lost their nourseothricin resistance upon subculturing in nonselective medium. Our work shows that WGT is an efficient strategy for rapidly generating and identifying superior alleles capable of improving selectable traits of interest in industrial yeast strains.

Spacer prioritization in CRISPR-Cas9 immunity is enabled by the leader RNA.

CRISPR-Cas systems store fragments of foreign DNA called spacers as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the newest spacers are known to be prioritized for immune defense. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR-Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defense against the matching invader. Using the CRISPR-Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes tracrRNA hybridization with the second repeat, accelerating crRNA processing. Accordingly, disrupting this structure reduces the abundance of the associated crRNA and immune defense against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR-Cas9 systems. CRISPR-Cas systems thus possess an RNA-based mechanism to prioritize defense against the most recently encountered invaders.

CRISPR-based antimicrobials to obstruct antibiotic-resistant and pathogenic bacteria.

The rapid emergence of antibiotic-resistant bacteria and the comparatively limited development of new antibacterial molecules pose a major threat to modern medicine by jeopardizing our ability to treat and prevent infections [1]. The overuse of antibiotics in both medical and agricultural settings has facilitated the emergence of multidrug-resistant (MDR) bacteria [2]. Furthermore, many antibiotics lack specificity, indiscriminately killing pathogenic and nonpathogenic bacteria and contributing to antibiotic-associated infections [3,4]. This highlights the critical need for novel therapeutics that circumvent existing modes of drug resistance while adding specificity [5]. Toward this goal, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are being explored as possible antimicrobials to target bacteria and/or antibiotic resistance genes in a sequence-specific manner. These promising targeted therapies are the focus of synthetic biology companies such as Locus Biosciences, Intellia Therapeutics, and Eligo Bioscience, to name a few. Although these technologies are mostly in the preclinical phase, the main goal is to provide precision antimicrobials for infectious diseases. Here, we discuss the present state of CRISPR antimicrobial research, with a specific focus on the biological differences between targeting plasmids versus chromosomes, the efficacy of CRISPR antimicrobials in infection and colonization models, and the various delivery mechanisms for these potential therapeutic tools. Sequence-specific targeting by CRISPR/Cas CRISPR/Cas systems are adaptive defense mechanisms against mobile genetic elements (MGEs). A key hallmark of these systems is the CRISPR array, a genomic locus comprised of unique spacers alternated by identical repeats [6]. Effector proteins function as interference molecules to silence foreign genetic elements [6]. During adaptation, some CRISPR types integrate a short fragment of foreign DNA into the CRISPR array, thereby providing a genetic memory of MGE invasion, referred to as a spacer (Fig 1). Transcription of the CRISPR array yields precursor CRISPR RNAs (pre-crRNAs) that are enzymatically processed into mature crRNAs (Fig 1). Upon invasion of bacteria by MGEs with complementary sequence, crRNAs guide effector proteins to these targets for enzymatic cleavage, ultimately causing sequence-specific elimination of the invading molecule [6] (Fig 1). Open in a separate window Fig 1 Mechanisms of action of CRISPR/Cas immunity. Left: Class I CRISPR/Cas systems (modeled by Type I system) utilize a multi-subunit complex, termed “Cascade,” as the effector machinery. Right: Class II CRISPR/Cas systems (modeled by Type II system) use a single effector protein (such as Cas9) for interference. Both classes also consist of spacer (diamonds) and repeat (squares) arrays. Top: During adaptation, the Cas1–Cas2 complex takes a sequence of the invading DNA and integrates it into the CRISPR array as a novel spacer. Center: In the next stage, termed “expression,” the CRISPR array is transcribed into pre-crRNAs that are further processed into mature interfering crRNAs. Bottom: During the interference stage, the mature crRNAs guide the Cas proteins to their DNA target. Upon binding of the crRNA to their cognate DNA target, the Cas protein generates a double-stranded DNA break in the target. Created with BioRender.com. Cascade, CRISPR-associated complex for antiviral defense; CRISPR/Cas, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein.

Chloroplast Genomes of the Green-Tide Forming Alga Ulva compressa: Comparative Chloroplast Genomics in the Genus Ulva (Ulvophyceae, Chlorophyta)

To understand the evolution of Ulva chloroplast genomes at intraspecific and interspecific levels, in this study, three complete chloroplast genomes of Ulva compressa Linnaeus were sequenced and compared with the available Ulva cpDNA data. Our comparative analyses unveiled many noticeable findings. First, genome size variations of Ulva cpDNAs at intraspecific and interspecific levels were mainly caused by differences in gain or loss of group I/II introns, integration of foreign DNA fragments, and content of non-coding intergenic spacer regions. Second, chloroplast genomes of U. compressa shared the same 100 conserved genes as other Ulva cpDNA, whereas Ulva flexuosa appears to be the only Ulva species with the minD gene retained in its cpDNA. Third, five types of group I introns, most of which carry a LAGLIDADG or GIY-YIG homing endonuclease, and three of group II introns, usually encoding a reverse transcriptase/maturase, were detected at 26 insertion sites of 14 host genes in the 23 Ulva chloroplast genomes, and many intron insertion-sites have been found for the first time in Chlorophyta. Fourth, one degenerate group II intron previously ignored has been detected in the infA genes of all Ulva species, but not in the closest neighbor, Pseudoneochloris marina, and the other chlorophycean taxa, indicating that it should be the result of an independent invasion event that occurred in a common ancestor of Ulva species. Finally, the seven U. compressa cpDNAs represented a novel gene order which was different from that of other Ulva cpDNAs. The structure of Ulva chloroplast genomes is not conserved, but remarkably plastic, due to multiple rearrangement events.

Establishment of genomic library technology mediated by non-homologous end joining mechanism in Yarrowia lipolytica.

Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits. The non-homologous end joining (NHEJ), which enables foreign DNA fragments to be randomly integrated into different chromosomal sites, shows prominent capability in genomic libraries construction. In this study, we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process, employment of defective Ura marker and optimization of iterative transformations, which enhanced genes integration efficiency by 4.67, 22.74 and 1.87 times, respectively. We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE, CrtB and CrtI, with 23.8 times higher production than rDNA integration through homologous recombination (HR). The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites, with the copy number up to 65 and 53, respectively, creating potential for further application of recombinase mediated genome rearrangement in Y. lipolytica. This work provides a high-efficient NHEJ-mediated genomic library technology, which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h. This novel technology also lays an excellent foundation for the development of other genetic technologies in Y. lipolytica.

A CRISPR-Cas9-integrase complex generates precise DNA fragments for genome integration.

CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9′s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.

DNA-free genome editing

<p indent=0mm>CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been extensively used for gene editing of many plant species, including model plants such as <italic>Arabidopsis</italic> and rice, and major crops such as maize and wheat, as well as woody plants like poplar, bamboo, apple and citrus, etc. This technique has not only significantly promoted basic research, but has also facilitated the agronomic traits improvement of economically important crops or trees. Typically, the CRISPR/Cas system required its integration and expression of exogenous DNA in the genome of host plants, which lead to the cleavage of site-specific DNA double-strand breaks, and introduced the genome modifications during the DNA repair process. However, the integration of CRISPR/Cas elements brings several concerns for commercialization and basic research. First, the inserted CRISPR/Cas cassette is constitutively expressed in plant cells, and may increase the chance of off-target changes, as well as make it difficult to evaluate the stability and penetrance of the initially observed phenotypes. Second, plants with a foreign gene integrated into its genome were regarded as classic genetically modified organisms, which may potentially lead to the escape of the gene into the environment, leading to legislation concerns and strict government policy regulation about such organisms. Therefore, one of the main challenges in using the CRISPR/Cas system in commercial agriculture is to improve the plant’s agronomic traits by genome editing, meanwhile obtaining transgene-free mutant plants with stable transmissible properties. DNA-free genome editing technology in plants is new and began in 2015, and since then great advances have been made on producing transgenic-free genome-edited plants. The exogenous CRISPR/Cas cassette and other transgenes could be eliminated by multiple approaches after the target gene has been edited. For example, the CRISPR/Cas expression cassette can be removed through genetic segregation for plants that are reproduced sexually, and the end products are CRISPR-edited but transgene-free. Transgene-free gene-edited plants may also be generated by non-transgenic approaches, such as transient expression of mRNA encoding Cas nuclease and guide RNA without DNA integration, preassembled ribonucleoprotein and sgRNA complex transfection, fluorescence marker-assisted transgene cassette selection and elimination, virus-mediated and nano-material mediated genome editing technologies. By using these methods, people can obtain the genome-edited end plant product with increased agronomic traits, while no additional foreign DNA fragments have been introduced into the plant genome. Until now, at least 14 transgene-free and genome-edited plant species have been generated using this technology. In this review, we have highlighted these achievements, including methods, applications and the possibilities of their commercialization in plant breeding. We also discuss the current problems in using this technology, such as very low efficiency, limited gene modification types and labor-consuming aspects. With the development of new plant cell transformation methods and the applications of new typical CRISPR/Cas systems, the potential strategies for improving this revolutionary technology in the future are also discussed.

Multiple Intraspecific Variations of Mitochondrial Genomes in the Green-Tide Forming Alga, Ulva compressa Linnaeus (Ulvophyceae, Chlorophyta)

To gain further insights into intraspecific evolution of Ulva mitochondrial genomes, the mitogenomes of three morphotypes of the green-tide forming alga, Ulva compressa Linnaeus from China and USA, were sequenced and compared with the available data from Ulvophyceae. The U. compressa mitogenomes displayed significantly variable size at intraspecific level ranging from 61,700 to &gt;66,587 bp, due to difference in acquisition of foreign DNA fragments, gain or loss of both group I and II introns, and non-coding intergenic spacer regions. The U. compressa mitogenomes harbored variable gene content ranging from 69 genes (including orfs) in Uco1 to 76 in Uco5, and contained different intron content from 4 introns in Uco3 and Uco4 to 7 in Uco1. A total of 63 genes and only two group IB introns (intron cox1-1107 and cox1-1125) were shared by these five mitogenomes. The U. compressa mitogenomes accumulated much more inverted repeat (IR) elements, ranging from 45 in Uco1 to 88 in Uco2, than that of the other Ulva species (3 - 34). A locally collinear block of eight genes (rps11-rps19-rps4-rpl16-trnR-trnQ-trnE-trnS) with the size of 3,631 bp has been inverted only in Uco1 indicating that the rearrangement event happened after its divergence from Uco2-5 and might be relate to a specific IR element. A majority of the common genes (≈76%) displayed high identity (&gt;98%) among these five mitogenomes, while some low values were observed in six genes mainly due to duplication and insertion/deletion mutations of small DNA fragments. Our study presented the first case of multiple intraspecific variations in ulvophycean mitogenomes, and indicated that the mitogenome will be a valuable tool for understanding the native or nonindigenous nature of the cosmopolitan Ulva species.