Large DNA Fragments Research Articles

Combinatorial-Hierarchical DNA Library Design Using the TeselaGen DESIGN Module with j5.

Modern DNA assembly techniques are known for their potential to link multiple large DNA fragments together into even larger constructs in single pot reactions that are easier to automate and work more reliably than traditional cloning methods. The simplicity of the chemistry is in contrast to the increased work needed to design optimal reactions that maximize DNA fragment reuse, minimize cost, and organize thousands of potential chemical reactions. Here we examine available DNA assembly methods and describe through example, the construction of a complex but not atypical combinatorial and hierarchical library using protocols that are generated automatically with the assistance of modern synthetic biology software.

DNA assembly technologies: a review

DNA assembly is the core technology of synthetic biology. With the development of synthetic biology, researchers have developed different DNA assembly technologies that rely on DNA polymerase or DNA ligase, and also have developed some non-enzyme-dependent DNA assembly techniques to facilitate the automation of DNA assembly. The assembly of large fragments of DNA from a few hundred kb to Mb is mostly dependent on microbial recombination. In this paper, the three types of DNA assembly technologies, including enzyme-dependent, non-enzymatic and in vivo homologous recombination, are reviewed.

Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction.

Background Choosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences. Methods We isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR. Results The amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts. Conclusions Our results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

Expression and purification of recombinant puroindoline A protein in Escherichia coli and its antifungal effect against Aspergillus flavus.

Aspergillus flavus is the main cause of postharvest agricultural commodity loss. In this study, puroindoline A (PINA) protein was expressed in Escherichia coli, purified, and its antifungal properties against A. flavus were characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the molecular weight of the recombinant PINA protein was approximately 44 kDa. PINA exerted a powerful antifungal effect against A. flavus at 42.42 μg/mL on potato dextrose agar culture medium. Flow cytometry and scanning electron microscopy revealed that the spore morphology was damaged by PINA exposure; spores were depressed and broken, suggesting that the cell wall was impaired. Transmission electron microscopy and propidium iodide staining illustrated significant changes in intracellular spore structure, indicating cell membrane damage. 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide staining indicated decreased mitochondrial membrane potential. Large nuclear condensation and DNA fragmentation were detected by 4',6-diamidino-2-phenylindole staining. The expression of genes related to the cell wall, cell membrane, and spore germination significantly changed in PINA-treated cells; this illustrated the probable mode of PINA action on A. flavus through cell wall destruction and triggered cell membrane, mitochondrial, and DNA damage leading to cell death. The antifungal mechanism of wheat PINA protein on A. flavus has been demonstrated in this study, and has potential application in preventing postharvest loss in the agricultural industry.

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum

Acremonium chrysogenum has been employed in the industrial production of cephalosporin C (CPC). However, there are still some impediments to understanding the regulation of CPC biosynthesis and improving strains due to the difficulty of genetic manipulation in A. chrysogenum, especially in the CPC high-producing strain C10. Here, an improved CRISPR-Cas9 system was constructed based on an U6/tRNA chimeric promoter. Using this system, high efficiency for single gene disruption was achieved in C10. In addition, double loci were simultaneously targeted when supplying with the homology-directed repair templates (donor DNAs). Based on this system, large DNA fragments up to 31.5 kb for the yellow compound sorbicillinoid biosynthesis were successfully deleted with high efficiency. Furthermore, CPC production was significantly enhanced when the sorbicillinoid biosynthetic genes were knocked out. This study provides a powerful tool for gene editing and strain improvement in A. chrysogenum.

ADRB2 Gene Knockout in Human Primary T Cells by Multiple sgRNAs Construced using CRISPR/Cas9 Technology

To knockout ADRB2 gene rapidly and efficiently in human primary T cells by using CRISPR/Cas9 technology and multiple sgRNAs strategy. Six paired-sgRNAs, which were designed to target the 5' constitutive coding exons of ADRB2 gene, were cloned into pGL3-U6-sgRNA-PGK-Puro vector separately. The expre-ssion vectors containing the single sgRNAs were constructed and transiently co-transfected into HEK-293T cell line with Cas9 expression vector. The sgRNA-mediated cleavage efficiency was tested by T7EN I digestion assay. Concatenating four highly efficient paired sgRNAs were cloned into pGL3-U6-sgRNA-ccdB-EF1α-Puro expression vector. The reco-mbinant plasmid allows the cells to express 4 sgRNAs, which target different sites on the ADRB2 genomic locus. The cleavage efficiency and mutation model were tested by T7EN I digest assay and T-A cloning technique. Multiple sgRNAs plasmid and Cas9 plasmid was transiently transferred into human primary T cells by electroporation. Flow cytometry (FCM) was used to detect the knockout efficiency of β2 adrenergic receptor (β2-AR). The results of T7EN I digestion and TA cloning sequencing showed that the multiple sgRNAs strategy could obtain more abundant mutation types and higher gene editing efficiency than single sgRNA. In addition to the deletion and insertion of bases, large fragment DNA deletions and inversions could be observed. All of the random 10 TA clones for detection were genetically modified, thus the mutation efficiency was as high as 100%. FCM assay showed that 43.09% of the cells in the control T cells were β2-AR positive, but the proportion of β2-AR positive cells in the multiple sgRNAs electrotransformed T cells decreased to 25.61%. A method, which is simple and operable, for knocking out β2-AR in human primary T cells has been established preliminarily. The results are helpful for the further study of the role of β2-AR in human T cells.

Characterization of the Noncanonical Regulatory and Transporter Genes in Atratumycin Biosynthesis and Production in a Heterologous Host.

Atratumycin is a cyclodepsipeptide with activity against Mycobacteria tuberculosis isolated from deep-sea derived Streptomyces atratus SCSIO ZH16NS-80S. Analysis of the atratumycin biosynthetic gene cluster (atr) revealed that its biosynthesis is regulated by multiple factors, including two LuxR regulatory genes (atr1 and atr2), two ABC transporter genes (atr29 and atr30) and one Streptomyces antibiotic regulatory gene (atr32). In this work, three regulatory and two transporter genes were unambiguously determined to provide positive, negative and self-protective roles during biosynthesis of atratumycin through bioinformatic analyses, gene inactivations and trans-complementation studies. Notably, an unusual Streptomyces antibiotic regulatory protein Atr32 was characterized as a negative regulator; the function of Atr32 is distinct from previous studies. Five over-expression mutant strains were constructed by rational application of the regulatory and transporter genes; the resulting strains produced significantly improved titers of atratumycin that were ca. 1.7–2.3 fold greater than wild-type (WT) producer. Furthermore, the atratumycin gene cluster was successfully expressed in Streptomyces coelicolor M1154, thus paving the way for the transfer and recombination of large DNA fragments. Overall, this finding sets the stage for understanding the unique biosynthesis of pharmaceutically important atratumycin and lays the foundation for generating anti-tuberculosis lead compounds possessing novel structures.

Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system for Bacillus subtilis

BackgroundMetabolic engineering has expanded from a focus on designs requiring a small number of genetic modifications to increasingly complex designs driven by advances in multiplex genome editing technologies. However, simultaneously modulating multiple genes on the chromosome remains challenging in Bacillus subtilis. Thus, developing an efficient and convenient method for B. subtilis multiplex genome editing is imperative.ResultsHere, we developed a CRISPR/Cas9n-based multiplex genome editing system for iterative genome editing in B. subtilis. This system enabled us to introduce various types of genomic modifications with more satisfying efficiency than using CRISPR/Cas9, especially in multiplex gene editing. Our system achieved at least 80% efficiency for 1–8 kb gene deletions, at least 90% efficiency for 1–2 kb gene insertions, near 100% efficiency for site-directed mutagenesis, 23.6% efficiency for large DNA fragment deletion and near 50% efficiency for three simultaneous point mutations. The efficiency for multiplex gene editing was further improved by regulating the nick repair mechanism mediated by ligD gene, which finally led to roughly 65% efficiency for introducing three point mutations on the chromosome. To demonstrate its potential, we applied our system to simultaneously fine-tune three genes in the riboflavin operon and significantly improved the production of riboflavin in a single cycle.ConclusionsWe present not only the iterative CRISPR/Cas9n system for B. subtilis but also the highest efficiency for simultaneous modulation of multiple genes on the chromosome in B. subtilis reported to date. We anticipate this CRISPR/Cas9n mediated system to greatly enhance the optimization of diverse biological systems via metabolic engineering and synthetic biology.

Pulsed-Field Gel Electrophoresis Typing of Staphylococcus aureus Strains.

Pulsed-field gel electrophoresis (PFGE) is one of the most effective genotyping methods for the study of genetic relatedness of microorganisms as well as for epidemiologic investigation of outbreaks caused by pathogenic bacteria, including Staphylococcus aureus. The technique relies on the size patterns of large DNA fragments generated by the cleavage of intact bacterial chromosomes with a rare cutting restriction enzyme, and subsequently resolved by pulsed-field electrophoresis with periodic changes of the orientation of the electrical field across the gel. The high discriminatory power, improved reproducibility by standardization of experimental protocols and data analysis guidelines, and establishment of a national PFGE database of S. aureus profiles have made it a valuable means for global tracking of S. aureus infection sources and determination of genetic relatedness of the strains.

An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters

Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable. Here, we use the facultative anaerobe Streptococcus mutans UA159 as a heterologous host for the expression of BGCs from anaerobic bacteria. A natural competence based large DNA fragment cloning (NabLC) technique was developed, which can move DNA fragments up to 40-kb directly and integrate a 73.7-kb BGC to the genome of S. mutans UA159 via three rounds of NabLC cloning. Using this system, we identify an anti-infiltration compound, mutanocyclin, from undefined BGCs from human oral bacteria. We anticipate this host system will be useful for heterologous expression of BGCs from anaerobic bacteria.

Characterization and phylogenomics of the complete mitochondrial genome of the polyzoic cestode Gangesia oligonchis (Platyhelminthes: Onchoproteocephalidea).

The order Onchoproteocephalidea (Eucestoda) was recently erected to accommodate the hook-bearing tetraphyllideans and the proteocephalideans, which are characterized by internal proglottization and a tetra-acetabulate scolex. The recognized subfamilies in the Proteocephalidae appeared to be non-monophyletic based on 28S recombinant DNA (rDNA) sequence data. Other molecular markers with higher phylogenetic resolution, such as large mitochondrial DNA fragments and multiple genes, are obviously needed. Thus the mitochondrial genome of Gangesia oligonchis, belonging to the putative earliest diverging group of the Proteocephalidae, was sequenced. The circular mitogenome of G. oligonchis was 13,958 bp in size, and contained the standard 36 genes: 22 transfer RNA genes, two rRNA genes and 12 protein-coding genes, as well as two major non-coding regions. A short NCR and a large NCR (lNCR) region were 216 bp and 419 bp in size, respectively. Highly repetitive regions in the lNCR region were detected with that of 11 repeat units. The mitogenome of G. oligonchis shared 71.1% nucleotide identity with Testudotaenia sp. WL-2016. Phylogenetic analyses of the complete mitochondrial genomes with Bayesian inference and maximum likelihood methods indicated that G. oligonchis formed a sister clade with Testudotaenia sp. WL-2016 with maximum support. The ordinal topology is (Caryophyllidea, (Diphyllobothriidea, (Bothriocephalidea, (Onchoproteocephalidea, Cyclophyllidea)))). The mitogenomic gene arrangement of G. oligonchis was identical to that of Testudotaenia sp. WL-2016. Both mitogenomic and nuclear sequence data for many more taxa are required to effectively explore the inter-relationships among the Onchoproteocephalidea.

Abstract 2239: Analysis of nucleosomal DNA as an extraction control for plasma-based circulating tumor DNA assays

Abstract Purpose: Challenges to accurate quantitative measurements of circulating tumor DNA (ctDNA) include variable contamination with large DNA fragments released during mononuclear cell lysis, and potential variability in extraction efficiency. We investigated optimal procedures for plasma processing and the potential utility of a DNA spike-in as a process control for extraction efficiency. Methods: Initial experiments analyzed the potential benefits of different centrifugation protocols (1600 x g vs. 1600 x g followed by 16000 x g) for plasma separation by analyzing the size distribution of extracted DNA using a BioAnalyzer. In subsequent experiments we used droplet digital PCR (ddPCR) to compare differences in extraction efficiency between nucleosomal DNA (nDNA) and genomic DNA (gDNA) spiked into healthy donor plasma and extracted using the QIAamp DSP Circulating NA Kit (Qiagen). To evaluate the properties of the purification columns with respect to the yield of different DNA sizes, we compared the plasma results to experiments extracting purified nDNA and gDNA spiked into PBS instead of plasma. We investigated the potential competitive effect of large DNA on the extraction efficiency of small DNA by mixing nDNA with commercially available human gDNA in different concentrations. To assess if nDNA, when spiked into patient plasma, would track with endogenous ctDNA, we spiked a pool of plasma from patients with BRAFV600E mutations with known quantities of nDNA purified from NRASQ61K mutant cells (CHP212). The spiked plasma pool was divided and extracted using multiple separate columns. Copies/ml and mutant fraction were determined by ddPCR assays for BRAFV600E and NRASQ61K. Coefficients of variation were also calculated. Results: Comparison of different centrifugation speeds for plasma processing before and after freeze-thaw indicated that a 2nd high-speed centrifugation (16000 x g), either before or after a freeze-thaw cycle, minimizes large DNA in plasma samples prior to ctDNA extraction. In initial spike-in experiments extraction efficiency for nDNA was 87.7% compared to 42% for gDNA. We obtained similar results when purifying the nDNA and gDNA from PBS instead of plasma. We observed greater variability in nDNA extraction efficiency when increasing amounts of gDNA were added as a contaminant. Finally, we found that the slight, paralell variations in extraction yields of externally spiked NRASQ61K mutant nDNA and endogenous BRAFV600E ctDNA in 7/9 replicate extractions. The overall coefficients of variation (total copies/mL) for 9 extractions for BRAFV600E and NRASQ61K were 3.9 and 4.2 respectively. Conclusion: A second centrifugation step helps reduce potential contamination of ctDNA with large DNA, and may improve the recovery of ctDNA from plasma samples. Nucleosomal DNA can potentially be used as a process control for extracting ctDNA from plasma samples. Citation Format: Mahrukh M. Syeda, George Karlin-Neumann, Iman Osman, David Polsky. Analysis of nucleosomal DNA as an extraction control for plasma-based circulating tumor DNA assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2239.

Abstract 1706: High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types

Abstract Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, whole-genome sequencing (WGS) of these samples has remained challenging due to the difficult process of isolating high-quality DNA and distinguishing true variant calls from artifacts. As evidence of these challenges, only four WGS studies of FFPE samples have been published to date. Previous work has attempted to overcome these challenges by using whole-exome sequencing or smaller exon panels at high coverage depths with publications reporting success from less than half to most samples. Thus, there has not been widespread adoption of using FFPE samples for next-generation sequencing. We developed a novel DNA extraction and library prep protocol for whole-genome sequencing of FFPE tissue samples. This method produces larger DNA fragments that can be successfully used for WGS. We previously presented results of a large esophageal cancer cohort where we sequenced over 1,000 samples in tumor-normal pairs with data equivalent to that generated from fresh-frozen tissues. We recently expanded the tumor types, ages, and sources and achieved similar results. We have assayed FFPE tissues derived from breast cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, and melanoma. These samples were sourced from several different labs, which have used variations on the standard fixation protocol. Some of these FFPE samples were over 15 years old. Variant calling provides excellent results without excessive C&amp;gt;T mutations. The coverage uniformity was very good, and CNV calls were made with results comparable to published TCGA data. These samples have been analyzed for MSI and TMB, and the results are comparable to those using fresh tissues. Our method makes new types of clinical studies possible utilizing FFPE samples that have been generated as part of standard hospital procedures. This method also facilitates performing studies comparing primary and recurrent cancers easier to establish and run as recurrent cancer cases can be enrolled and stored FFPE primary samples used to establish a cancer baseline. Citation Format: Shannon T. Bailey, James Lund, Muhammad Ekram, Wanfeng Yu, Richard T. Williams, Hongye Sun, Jeffrey R. Gulcher. High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1706.

Abstract 3529: Sample quality control of cell-free DNA

Abstract Sequencing of cell-free DNA (cfDNA) is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on pre-analytical sample treatment or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of an automated electrophoresis platform performing cfDNA quantification with region analysis. Moreover, the results include a score to qualify cfDNA samples according to their contamination level with high molecular weight material. This allows defining a threshold for objective sample qualification prior to library preparation. The analysis features are described with examples of typical sample patterns. Citation Format: Eva Graf. Sample quality control of cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3529.

Endogenous Expression of the Human CD83 Attenuates EAE Symptoms in Humanized Transgenic Mice and Increases the Activity of Regulatory T Cells.

The CD83 is a type I membrane protein and part of the immunoglobulin superfamily of receptors. CD83 is involved in the regulation of antigen presentation and dendritic cell dependent allogeneic T cell proliferation. A soluble form of CD83 inhibits dendritic cell maturation and function. Furthermore, CD83 is expressed on activated B cells, T cells, and in particular on regulatory T cells. Previous studies on murine CD83 demonstrated this molecule to be involved in several immune-regulatory processes, comprising that CD83 plays a key role in the development und function of different immune cells. In order to get further insights into the function of the human CD83 and to provide preclinical tools to guide the function of CD83/sCD83 for therapeutic purposes we generated Bacterial Artificial Chromosomes (BAC) transgenic mice. BACs are excellent tools for manipulating large DNA fragments and are utilized to engineer transgenic mice by pronuclear injection. Two different founders of BAC transgenic mice expressing human CD83 (BAC-hCD83tg mice) were generated and were examined for the hCD83 expression on different immune cells as well as both the in vitro and in vivo role of human CD83 (hCD83) in health and disease. Here, we found the hCD83 molecule to be present on activated DCs, B cells and subtypes of CD4+ T cells. CD8+ T cells, on the other hand, showed almost no hCD83 expression. To address the function of hCD83, we performed in vitro mixed lymphocyte reactions (MLR) as well as suppression assays and we used the in vivo model of experimental autoimmune encephalomyelitis (EAE) comparing wild-type and hCD83-BAC mice. Results herein showed a clearly diminished capacity of hCD83-BAC-derived T cells to proliferate accompanied by an enhanced activation and suppressive activity of hCD83-BAC-derived Tregs. Furthermore, hCD83-BAC mice were found to recover faster from EAE-associated symptoms than wild-type mice, encouraging the relevance also of the hCD83 as a key molecule for the regulatory phenotype of Tregs in vitro and in vivo.

Large Enriched Fragment Targeted Sequencing (LEFT-SEQ) Applied to Capture of Wolbachia Genomes

Symbiosis is a major force of evolutionary change, influencing virtually all aspects of biology, from population ecology and evolution to genomics and molecular/biochemical mechanisms of development and reproduction. A remarkable example is Wolbachia endobacteria, present in some parasitic nematodes and many arthropod species. Acquisition of genomic data from diverse Wolbachia clades will aid in the elucidation of the different symbiotic mechanisms(s). However, challenges of de novo assembly of Wolbachia genomes include the presence in the sample of host DNA: nematode/vertebrate or insect. We designed biotinylated probes to capture large fragments of Wolbachia DNA for sequencing using PacBio technology (LEFT-SEQ: Large Enriched Fragment Targeted Sequencing). LEFT-SEQ was used to capture and sequence four Wolbachia genomes: the filarial nematode Brugia malayi, wBm, (21-fold enrichment), Drosophila mauritiana flies (2 isolates), wMau (11-fold enrichment), and Aedes albopictus mosquitoes, wAlbB (200-fold enrichment). LEFT-SEQ resulted in complete genomes for wBm and for wMau. For wBm, 18 single-nucleotide polymorphisms (SNPs), relative to the wBm reference, were identified and confirmed by PCR. A limit of LEFT-SEQ is illustrated by the wAlbB genome, characterized by a very high level of insertion sequences elements (ISs) and DNA repeats, for which only a 20-contig draft assembly was achieved.

Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in

The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9’s efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.

Lateral transfers of large DNA fragments spread functional genes among grasses

A fundamental tenet of multicellular eukaryotic evolution is that vertical inheritance is paramount, with natural selection acting on genetic variants transferred from parents to offspring. This lineal process means that an organism's adaptive potential can be restricted by its evolutionary history, the amount of standing genetic variation, and its mutation rate. Lateral gene transfer (LGT) theoretically provides a mechanism to bypass many of these limitations, but the evolutionary importance and frequency of this process in multicellular eukaryotes, such as plants, remains debated. We address this issue by assembling a chromosome-level genome for the grass Alloteropsis semialata, a species surmised to exhibit two LGTs, and screen it for other grass-to-grass LGTs using genomic data from 146 other grass species. Through stringent phylogenomic analyses, we discovered 57 additional LGTs in the A. semialata nuclear genome, involving at least nine different donor species. The LGTs are clustered in 23 laterally acquired genomic fragments that are up to 170 kb long and have accumulated during the diversification of Alloteropsis. The majority of the 59 LGTs in A. semialata are expressed, and we show that they have added functions to the recipient genome. Functional LGTs were further detected in the genomes of five other grass species, demonstrating that this process is likely widespread in this globally important group of plants. LGT therefore appears to represent a potent evolutionary force capable of spreading functional genes among distantly related grass species.

CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum

Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, we developed a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The HITI strategy was able to facilitate integration of a large DNA fragment, ∼8 kb in length, into the genome of F. oxysporum at the sgRNA cleavage site, and was used to insert a C-terminal 3×sGFP tag to the FoCHS5 gene and a N-terminal mCherry tag to the FoSSO2 gene. The HDRI strategy was used to tag the paralogous gene, FoSSO1, with a C-terminal mCherry marker. FoChs5-3×sGFP localized to conidia, some septa, and fungal tips. A majority of the FoSso1-mCherry was distributed in the conidia, septum, and hyphae that were distal from the fungal tips. While FoSso1-mCherry showed a very weak fluorescent signal at the fungal tips, mCherry-FoSso2 accumulated in the plasma membrane of conidia, germlings, fungal tips, hyphae, and phialides, suggesting FoSSO1 and FoSSO2 are regulated differently during fungal development. These results indicate this EGT system is efficient and can be another molecular tool to resolve the function(s) of proteins and infection strategies of F. oxysporum.