Efficiency Of Mutagenesis Research Articles

Analysis of FR901379 Biosynthetic Genes in Coleophoma empetri by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Based Genomic Manipulation.

The compound FR901379, a sulfated echinocandin produced by the filamentous fungus Coleophoma empetri F-11899, is an important intermediate for the synthesis of the antifungal drug micafungin. In this study, we established an efficient clustered regularly interspaced short palindromic repeats/Cas9-based gene editing tool for the industrial production strain C. empetri SIPI1284. With this method, the efficiency of gene mutagenesis in the target locus is up to 84%, which enables the rapid gene disruption for the analysis of FR901379 biosynthetic genes. Next, we verified the putative functional genes of the FR901379 biosynthetic gene cluster via gene disruption and gene complementation in vivo. These core functional genes included the nonribosomal peptide synthetase gene (CEnrps), the fatty-acyl-AMP ligase gene (CEligase) responsible for the formation of the activated form of palmitic acid and its transfer to CEnrps, four nonheme mononuclear iron oxygenase genes (CEoxy1, CEoxy2, CEoxy3, and CEoxy4) responsible for the synthesis of nonproteinogenic amino acids, l-homotyrosine biosynthesis genes (CEhtyA-D), two cytochrome P450 enzyme genes (CEp450-1 and CEp450-2), and a transcription regulator gene (CEhyp). In addition, by screening the whole genome, we identified two unknown genes (CEp450-3 and CEsul) responsible for the sulfonyloxy group of FR901379, which were separated from the core FR901379 biosynthetic cluster. Furthermore, during gene disruptions in the research, we obtained a series of FR901379 analogues and elucidated the relationship between the groups and antifungal activities.

Cas9-induced large deletions and small indels are controlled in a convergent fashion

Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.

Epigenetic features drastically impact CRISPR-Cas9 efficacy in plants.

CRISPR–Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR–Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR–Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR–Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR–Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.

Enhanced Codon-Anticodon Interaction at In-Frame UAG Stop Codon Improves the Efficiency of Non-Natural Amino Acid Mutagenesis.

The introduction of non-natural amino acids into proteins through the stop codon readthrough methodology has been used to design proteins for diverse applications. However, this method suffers from low yields of the modified protein, as the suppressor tRNA that recognizes the stop codon is unable to compete effectively with release factor 1 (RF1), which terminates translation. We reasoned that a suppressor tRNA with improved interaction with the UAG stop codon on the mRNA will be able to compete more effectively with RF1. To test this idea, we inserted two 2,6-diaminopurine (D) units in the tRNA anticodon stem loop, including one at the third position of the tRNA anticodon. The modified suppressor tRNA could potentially form additional H-bonds between the N2-exocyclic amine of D and the C2 carbonyl group of uracil, thereby enhancing mRNA-tRNA interaction and/or altering tRNA conformation. The stronger interaction at the codon-anticodon interface resulted in improved UAG decoding efficiency and a higher yield of the modified protein containing a non-natural amino acid at multiple sites. Our findings are consistent with the importance of hydrogen bonding and tRNA conformation at the tRNA-mRNA duplex interface during in-frame UAG suppression, which improves protein translation at multiple UAG stop sites. This work provides valuable inputs toward improved non-natural amino acid mutagenesis for creating designer proteins.

The heat is on: a simple method to increase genome editing efficiency in plants

BackgroundPrecision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress.ResultsHere, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions.ConclusionsTogether, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.

Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants.

Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7–41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0–85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.

A High-Throughput Strategy for T-Cell Receptor Cloning and Expression.

Expression of T-cell receptor (TCR) genes is a critical step for TCR characterization and epitope identification. The recent interest in using specific TCRs for cancer immunotherapy has further increased the demand for practical and robust methods to rapidly clone and express TCRs. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TCR transgene into different expression systems. In this protocol, we first constructed all the human TRAV and TRBV genes into individual plasmid. To clone any TCR, we only need to ligate a short CDR3 fragment to its corresponding V gene plasmid using Golden Gate cloning. This strategy significantly improves the efficiency of individual TCR cloning and mutagenesis, providing a flexible high-throughput method for TCR analysis and TCR-mediated therapeutics.

Application of Protoplast Regeneration to CRISPR/Cas9 Mutagenesis in Nicotiana tabacum.

Protoplast transfection is widely used in plant research to rapidly evaluate RNA degradation, reporter assay, gene expression, subcellular localization, and protein-protein interactions. In order to successfully use protoplast transfection with the newly emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein editing platform, high yield of protoplasts, stable transfection efficiency, and reliable regeneration protocols are necessary. The Nicotiana tabacum transient protoplast transfection and regeneration system can effectively obtain target gene mutations in regenerated plants without transgenes and is thus a very attractive technique for evaluating gene editing reagents using CRISPR/Cas-based systems. Here, we describe in detail sterilized seed germination, culture conditions, isolation of Nicotiana tabacum protoplasts from tissue culture explants, construction of a vector containing the Cas protein and sgRNA cassette, highly efficient polyethylene glycol-calcium transient transfection of plasmids delivered into protoplasts, evaluation of mutagenesis efficiency and genotype analysis from protoplasts and regenerated plants, and the regeneration conditions to obtain CRISPR-edited plants from single protoplasts.

An Efficient Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Protein 9 Mutagenesis System for Oil Palm (Elaeis guineensis).

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful tool for the precise editing of plant genomes for crop improvement. Rapid in vitro methods for the determination of guide RNA (gRNA) cleavage efficiency and an efficient DNA delivery system is essential for gene editing. However, we lack an efficient gene-editing system for palm species. In this study, we described the development of a transient oil palm protoplast assay to rapidly evaluate the cleavage efficiency of CRISPR/Cas9 mutagenesis and the generation of stable transformed oil palms using biolistic particle bombardment in immature embryos. Using the phytoene desaturase (EgPDS) gene, we found cleavage frequency of up to 25.49% in electro-transfected protoplast, which enables the production of transgenic oil palm shoots exhibiting chimeric albino phenotypes as a result of DNA insertions, deletions (InDels), and nucleotide substitutions, with a mutation efficiency of 62.5–83.33%. We further validated the mutagenesis efficiency and specificity of the CRISPR/Cas9 system in oil palm by targeting the brassinosteroid-insensitive 1 (EgBRI1) gene, which resulted in nucleotide substitutions in EgBRI1 with premature necrosis phenotype in oil palm transgenic shoots and stunted phenotype resulting from DNA InDels. Taken together, our results showed that effective and efficient editing of genes using the CRISPR/Cas9 system can be achieved in oil palm by optimizing the selection of efficient gRNA and DNA delivery methods. This newly designed strategy will enable new routes for the genetic improvement in oil palm and related species.

Creation of a potato mutant lacking the starch branching enzyme gene StSBE3 that was generated by genome editing using the CRISPR/dMac3-Cas9 system.

The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.

Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application.

In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.

Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

Efforts to improve CRISPR-Cas9 genome editing systems for lower off-target effects are mostly at the cost of its robust on-target efficiency. To enhance both accuracy and efficiency, we created chimeric SpyCas9 proteins fused with the 5′-to-3′ exonuclease Recombination J (RecJ) or with GFP and demonstrated that transfection of the pre-assembled ribonucleoprotein of the two chimeric proteins into human or plant cells resulted in greater targeted mutagenesis efficiency up to 600% without noticeable increase in off-target effects. Improved activity of the two fusion proteins should enable editing of the previously hard-to-edit genes and thus readily obtaining the cells with designer traits.

Genome Editing Using the CRISPR-Cas9 System to Generate a Solid-Red Germline of Nile Tilapia (Oreochromis niloticus).

In recent years, there has been increasing demand for red tilapia, which are commercial strains of hybrids of different tilapiine species or red variants of highly inbred Nile tilapia. However, red tilapia phenotypes are genetically unstable and affected by environmental factors, resulting in nonuniform coloration with black or dark-red color blotches that reduce their market value. Solute carrier family 45 member 2 (SLC45A2) is a membrane transporter that mediates melanin biosynthesis and is evolutionarily conserved from fish to humans. In the present study, we describe the generation of a stable and heritable red tilapia phenotype by inducing loss-of-function mutations in the slc45a2 gene. For this purpose, we identified the slc45a2 gene in Nile tilapia and designed highly specific guide RNAs (gRNA) for its genomic sequence. Multiplex microinjection of slc45a2-specific ribonucleoproteins to Nile tilapia zygotes induced up to 97-99% albinism, including loss of melanin in the eye. Next-generation sequencing of the injected zygotes demonstrated that all injected fish carried mutant alleles with variable mutagenesis efficiencies. Sanger sequencing of the genomic target region in the slc45a2 gene from fin clips, sperm, and F1 offspring of a highly mutant male identified various genomic indels and germline transmission of the sperm-identified indels. Overall, this work demonstrates the generation of somatic and germline slc45a2 mutant alleles, which leads to complete albinism in Nile tilapia.

Effect of Three Types of Ion Beam Irradiation on Gerbera (Gerbera hybrida) In Vitro Shoots with Mutagenesis Efficiency.

Gerbera in vitro shoots were irradiated using three types of ion beams with different line energy transfers (LETs) to investigate the effective LET and absorbed doses for mutagenesis. Furthermore, genomic mutation analyses were conducted on the obtained mutants. Survival rate analysis showed a lower lethal dose 50% (LD50) with ion beams with higher LETs. Trait/morphological mutations exhibited changes in the color and shape of petals and male sterility. Irradiation conditions with the highest growth change and trait/morphological mutation rates in each ion were C irradiation at 10 Gy, Ar irradiation at 5 Gy, and Fe irradiation at 5 Gy, with a range of absorbed dose of around LD50 to about 10 Gy lower. The highest trait/morphological mutation rate was 14.1% with Ar irradiation at 5 Gy, which was one of the criteria for ion beam irradiation of gerbera in vitro shoots. Furthermore, the genomic mutation in the flower color, petal shape, and male sterile mutants were confirmed by genotype analysis using Genotyping by Random Amplicon Sequencing-Direct technology. This is the first study to report the efficient production of gerbera mutants that could be analyzed. Our findings may lead to more efficient gerbera mutant production and analysis technology.

CRISPR–Cas9 Mutagenesis in Xenopus tropicalis for Phenotypic Analyses in the F0 Generation and Beyond

CRISPR-Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid frog Xenopus tropicalis Here we describe a simple mutagenesis protocol using microinjection of Cas9 protein or mRNA, together with synthetic guide RNAs (sgRNAs) targeting specific DNA sequences, into the early embryo. Cas9-catalyzed double-strand breaks undergo error-prone repair, resulting in production of short insertions and/or deletions. Thus, careful selection of target sites can lead to mutations that impair normal function of the protein product. CRISPR-Cas9 can be used to create either mosaic loss-of-function Xenopus embryos that display F0 generation phenotypes or mutant lines for downstream analysis. In addition to describing how to mutagenize genes using CRISPR-Cas9, we also discuss a simple method to determine the mutagenesis efficiency, some potential problems that can arise, and possible solutions to overcome them. The protocol described here should be applicable to other amphibians and, in principle, many other organisms.

A transgenic system for targeted ablation of reproductive and maternal-effect genes.

Maternally provided gene products regulate the earliest events of embryonic life, including formation of the oocyte that will develop into an egg, and eventually into an embryo. Forward genetic screens have provided invaluable insights into the molecular regulation of embryonic development, including the essential contributions of some genes whose products must be provided to the transcriptionally silent early embryo for normal embryogenesis, called maternal-effect genes. However, other maternal-effect genes are not accessible due to their essential zygotic functions during embryonic development. Identifying these regulators is essential to fill the large gaps in our understanding of the mechanisms and molecular pathways contributing to fertility and to maternally regulated developmental processes. To identify these maternal factors, it is necessary to bypass the earlier requirement for these genes so that their potential later functions can be investigated. Here, we report reverse genetic systems to identify genes with essential roles in zebrafish reproductive and maternal-effect processes. As proof of principle and to assess the efficiency and robustness of mutagenesis, we used these transgenic systems to disrupt two genes with known maternal-effect functions: kif5ba and bucky ball.

CRISPR/Cas9-mediated mutagenesis reveals the roles of calaxin in gastropod larval cilia.

Obtaining detectable knockout phenotypes in the G0 generation is essential for gene function studies. Although CRISPR/Cas9-mediated gene editing has been employed to knock out molluscan genes, detectable phenotypes in the G0 generation have not been reported in these animals. In this study, we determined the knockout phenotype of a cilium-related gene, calaxin, using CRISPR/Cas9 technology in the gastropod mollusk Lottia goshimai. Injections with the Cas9-sgRNA complex caused approximately 30-80% of the injected larvae to exhibit a short-cilia phenotype characteristic of shortened cilia and decreased motility in the larvae. This phenotype was detectable in the G0 generation and was consistent for two independent sgRNAs. Genotyping of the injected larvae revealed various types of deletions and insertions in the target gene, which occurred in all sequences from the short-cilia larvae. This result indicated that the short-cilia phenotype was indeed caused by calaxin knockout. This possibility was supported by an RNAi assay targeting calaxin, which produced a highly similar short-cilia phenotype. We observed that a single SNP in the target sequences of the sgRNAs could show varied effects on the efficiency of mutagenesis. These results help to establish a foundation for future studies on molluscan gene editing using the CRISPR/Cas9 technique and contribute to the body of knowledge on molluscan ciliary functions.

The Genome-Wide EMS Mutagenesis Bias Correlates With Sequence Context and Chromatin Structure in Rice.

Ethyl methanesulfonate (EMS) is a chemical mutagen believed to mainly induce G/C to A/T transitions randomly in plant genomes. However, mutant screening for phenotypes often gets multiple alleles for one gene but no mutant for other genes. We investigated the potential EMS mutagenesis bias and the possible correlations with sequence context and chromatin structure using the whole genome resequencing data collected from 52 rice EMS mutants. We defined the EMS-induced single nucleotide polymorphic sites (SNPs) and explored the genomic factors associated with EMS mutagenesis bias. Compared with natural SNPs presented in the Rice3K project, EMS showed a preference on G/C sites with flanking sequences also higher in GC contents. The composition of local dinucleotides and trinucleotides was also associated with the efficiency of EMS mutagenesis. The biased distribution of EMS-induced SNPs was positively correlated with CpG numbers, transposable element contents, and repressive epigenetic markers but negatively with gene expression, the euchromatin marker DNase I hypersensitive sites, and active epigenetic markers, suggesting that sequence context and chromatin structure might correlate with the efficiency of EMS mutagenesis. Exploring the genome-wide features of EMS mutagenesis and correlations with epigenetic modifications will help in the understanding of DNA repair mechanism.

Intricacies of conditional genetics in vascular biology.

Conditional or inducible recombinase-based genetics is still the gold standard to analyse gene function, given its high specificity, temporal control, limited toxicity and the many available genetic tools. However, it is based on methods that have inherent limitations and shortcomings. The purpose of this review is to summarize and contrast the different available methods used to perform conditional gene function analysis to better inform the community about their particularities and the need to use better methods. As any other biomedical field, the vascular biology field has moved from using and analysing standard gene knockout (KO) mice, to use conditional genetics to delete a given gene only at a given time point, cell-type or organ of interest. This is the only way to accurately understand a gene function and avoid other confounding factors. Therefore, nowadays the majority of laboratories working with mice use CreERT2-tamoxifen-inducible genetics. However, this necessary transition from the relatively simple KO genetics to the more sophisticated conditional genetics brought a series of additional methodological issues that are often overlooked or unappreciated. Recent findings from several laboratories have shown how important is to know what to expect from and control for in conditional genetics. Without this a priori knowledge, the quality, robustness, time and costs of conditional genetic experiments can be significantly compromised. We start this review by discussing the intricacies of the most simple and widely used methods to perform conditional genetics and then extend on the need of novel and more advanced methods to increase the ease, efficiency and reliability of conditional mutagenesis and gene function analysis.

Improving CRISPR/Cas9 mutagenesis efficiency by delaying the early development of zebrafish embryos

CRISPR/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40 min. Here we explored two options to improve CRISPR/Cas9 mutagenesis in zebrafish—extending the single-cell stage and defining conditions for carrying out mutagenesis in oocytes prior to in vitro fertilization. Previous work has shown that ovarian fluid from North American salmon species (coho and chinook salmon) prolong oocyte survival ex vivo so that they are viable for hours instead of dying within minutes if left untreated. We found that commonly farmed rainbow trout (Oncorhynchus mykiss) ovarian fluid (RTOF) has similar effect on zebrafish oocyte viability. In order to prolong single-cell stage, we incubated zebrafish zygotes in hydrogen sulfide (H2S) and RTOF but failed to see any effect. However, the reduction of temperature from standard 28 to 12 °C postponed the first cell division by about an hour. In addition, the reduction in temperature was associated with increased CRISPR/Cas9 mutagenesis rate. These results suggest that the easily applicable reduction in temperature facilitates CRISPR/Cas9 mutagenesis in zebrafish.