Systemic delivery of engineered compact AsCas12f by a positive-strand RNA virus vector enables highly efficient targeted mutagenesis in plants.

Because virus vectors can spread systemically autonomously, they are powerful vehicles with which to deliver genome-editing tools into plant cells. Indeed, a vector based on a positive-strand RNA virus, potato virus X (PVX), harboring SpCas9 and its single guide RNA (sgRNA), achieved targeted mutagenesis in inoculated leaves of Nicotiana benthamiana. However, the large size of the SpCas9 gene makes it unstable in the PVX vector, hampering the introduction of mutations in systemic leaves. Smaller Cas variants are promising tools for virus vector-mediated genome editing; however, they exhibit far lower nuclease activity than SpCas9. Recently, AsCas12f, one of the smallest known Cas proteins so far (one-third the size of SpCas9), was engineered to improve genome-editing activity dramatically. Here, we first confirmed that engineered AsCas12f variants including I123Y/D195K/D208R/V232A exhibited enhanced genome-editing frequencies in rice. Then, a PVX vector harboring this AsCas12f variant was inoculated into N. benthamiana leaves by agroinfiltration. Remarkably, and unlike with PVX-SpCas9, highly efficient genome editing was achieved, not only in PVX-AsCas12f-inoculated leaves but also in leaves above the inoculated leaf (fourth to sixth upper leaves). Moreover, genome-edited shoots regenerated from systemic leaves were obtained at a rate of >60%, enabling foreign DNA-free genome editing. Taken together, our results demonstrate that AsCas12f is small enough to be maintained in the PVX vector during systemic infection in N. benthamiana and that engineered AsCas12f offers advantages over SpCas9 for plant genome editing using virus vectors.

An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat.

BACKGROUND: CRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community. RESULTS: We identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately. CONCLUSION: Based on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.

Rational Design and Screening of Synthetic Promoters in Chlamydomonas reinhardtii.

Synthetic promoters are powerful tools to boost the biotechnological potential of microalgae as eco-sustainable industrial hosts. The increasing availability of transcriptome data on microalgae in a variety of environmental conditions allows to identify cis-regulatory elements (CREs) that are responsible for the transcriptional output. Furthermore, advanced cloning technologies, such as golden gate-based MoClo toolkits, enable the creation of modular constructs for testing multiple promoters and a range of reporter systems in a convenient manner. In this chapter, we will describe how to introduce in silico-identified CREs into promoter sequences, and how to clone the modified promoters into MoClo compatible vectors. We will then describe how these promoters can be evaluated and screened for transgene expression in an established microalgal model for genetic perturbation, i.e., Chlamydomonas reinhardtii.

The Improved Antineoplastic Activity of Thermophilic L-Asparaginase Tli10209 via Site-Directed Mutagenesis.

Amino acid deprivation therapy (AADT) is a novel anticancer therapy, considered nontoxic and selective. Thermophilic L-asparaginase enzymes display high stability and activity at elevated temperatures. However, they are of limited use in clinical applications because of their low substrate affinity and reduced activity under physiological conditions, which may necessitate an improved dosage, leading to side effects and greater costs. Thus, in an attempt to improve the activity of L-Asn at 37 °C, with the use of a semi-rational design, eight active-site mutants of Thermococcus litoralis DSM 5473 L-asparaginase Tli10209 were developed. T70A exhibited a 5.11-fold increase compared with the wild enzyme in physiological conditions. Double-mutant enzymes were created by combining mutants with higher hydrolysis activity. T70A/F36Y, T70A/K48L, and T70A/D50G were enhanced by 5.59-, 6.38-, and 5.58-fold. The immobilized enzyme applied in MCF-7 breast cancer cells only required one-seventh of the dose of the free enzyme to achieve the same inhibition rate under near-infrared irradiation. This provides a proof of concept that it is possible to reduce the consumption of L-Asn by improving its activity, thus providing a method to manage side effects.

A CRISPR-Cas-associated transposon system for genome editing in Burkholderia cepacia complex species.

Genome editing in non-model bacteria is important to understand gene-to-function links that may differ from those of model microorganisms. Although species of the Burkholderia cepacia complex (Bcc) have great biotechnological capacities, the limited genetic tools available to understand and mitigate their pathogenic potential hamper their utilization in industrial applications. To broaden the genetic tools available for Bcc species, we developed RhaCAST, a targeted DNA insertion platform based on a CRISPR-associated transposase driven by a rhamnose-inducible promoter. We demonstrated the utility of the system for targeted insertional mutagenesis in the Bcc strains B. cenocepacia K56-2 and Burkholderia multivorans ATCC17616. We showed that the RhaCAST system can be used for loss- and gain-of-function applications. Importantly, the selection marker could be excised and reused to allow iterative genetic manipulation. The RhaCAST system is faster, easier, and more adaptable than previous insertional mutagenesis tools available for Bcc species and may be used to disrupt pathogenicity elements and insert relevant genetic modules, enabling Bcc biotechnological applications. IMPORTANCE: Species of the Burkholderia cepacia complex (Bcc) have great biotechnological potential but are also opportunistic pathogens. Genetic manipulation of Bcc species is necessary to understand gene-to-function links. However, limited genetic tools are available to manipulate Bcc, hindering our understanding of their pathogenic traits and their potential in biotechnological applications. We developed a genetic tool based on CRISPR-associated transposase to increase the genetic tools available for Bcc species. The genetic tool we developed in this study can be used for loss and gain of function in Bcc species. The significance of our work is in expanding currently available tools to manipulate Bcc.

Mutagenesis techniques for evolutionary engineering of microbes - exploiting CRISPR-Cas, oligonucleotides, recombinases, and polymerases.

The natural process of evolutionary adaptation is often exploited as a powerful tool to obtain microbes with desirable traits. For industrial microbes, evolutionary engineering is often used to generate variants that show increased yields or resistance to stressful industrial environments, thus obtaining superior microbial cell factories. However, even in large populations, the natural supply of beneficial mutations is typically low, which implies that obtaining improved microbes is often time-consuming and inefficient. To overcome this limitation, different techniques have been developed that boost mutation rates. While some of these methods simply increase the overall mutation rate across a genome, others use recent developments in DNA synthesis, synthetic biology, and CRISPR-Cas techniques to control the type and location of mutations. This review summarizes the most important recent developments and methods in the field of evolutionary engineering in model microorganisms. It discusses how both in vitro and in vivo approaches can increase the genetic diversity of the host, with a special emphasis on in vivo techniques for the optimization of metabolic pathways for precision fermentation.

Ultrahigh-throughput screening-assisted in vivo directed evolution for enzyme engineering.

BACKGROUND: Classical directed evolution is a powerful approach for engineering biomolecules with improved or novel functions. However, it traditionally relies on labour- and time-intensive iterative cycles, due in part to the need for multiple molecular biology steps, including DNA transformation, and limited screening throughput. RESULTS: In this study, we present an ultrahigh throughput in vivo continuous directed evolution system with thermosensitive inducible tunability, which is based on error-prone DNA polymerase expression modulated by engineered thermal-responsive repressor cI857, and genomic MutS mutant with temperature-sensitive defect for fixation of mutations in Escherichia coli. We demonstrated the success of the in vivo evolution platform with ß-lactamase as a model, with an approximately 600-fold increase in the targeted mutation rate. Furthermore, the platform was combined with ultrahigh-throughput screening methods and employed to evolve α-amylase and the resveratrol biosynthetic pathway. After iterative rounds of enrichment, a mutant with a 48.3% improvement in α-amylase activity was identified via microfluidic droplet screening. In addition, when coupled with an in vivo biosensor in the resveratrol biosynthetic pathway, a variant with 1.7-fold higher resveratrol production was selected by fluorescence-activated cell sorting. CONCLUSIONS: In this study, thermal-responsive targeted mutagenesis coupled with ultrahigh-throughput screening was developed for the rapid evolution of enzymes and biosynthetic pathways.

Multi-allelic gene editing in an apomictic, tetraploid turf and forage grass (Paspalum notatum Flüggé) using CRISPR/Cas9.

Polyploidy is common among grasses (Poaceae) and poses challenges for conventional breeding. Genome editing technology circumvents crossing and selfing, enabling targeted modifications to multiple gene copies in a single generation while maintaining the heterozygous context of many polyploid genomes. Bahiagrass (Paspalum notatum Flüggé; 2n=4x=40) is an apomictic, tetraploid C4 species that is widely grown in the southeastern United States as forage in beef cattle production and utility turf. The chlorophyll biosynthesis gene magnesium chelatase (MgCh) was selected as a rapid readout target for establishing genome editing in tetraploid bahiagrass. Vectors containing sgRNAs, Cas9 and nptII were delivered to callus cultures by biolistics. Edited plants were characterized through PCR-based assays and DNA sequencing, and mutagenesis frequencies as high as 99% of Illumina reads were observed. Sequencing of wild type (WT) bahiagrass revealed a high level of sequence variation in MgCh likely due to the presence of at least two copies with possibly eight different alleles, including pseudogenes. MgCh mutants exhibited visible chlorophyll depletion with up to 82% reductions in leaf greenness. Two lines displayed progression of editing over time which was linked to somatic editing. Apomictic progeny of a chimeric MgCh editing event were obtained and allowed identification of uniformly edited progeny plants among a range of chlorophyll depletion phenotypes. Sanger sequencing of a highly edited mutant revealed elevated frequency of a WT allele, probably due to frequent homology-directed repair (HDR). To our knowledge these experiments comprise the first report of genome editing applied in perennial, warm-season turf or forage grasses. This technology will accelerate bahiagrass cultivar development.

Multiplexed Guide RNA Expression Leads to Increased Mutation Frequency in Targeted Window Using a CRISPR-Guided Error-Prone DNA Polymerase in Saccharomyces cerevisiae.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, with its ability to target a specific DNA locus using guide RNAs (gRNAs), is particularly suited for targeted mutagenesis. The targeted diversification of nucleotides in Saccharomyces cerevisiae using a CRISPR-guided error-prone DNA polymerase─called yEvolvR─was recently reported. Here, we investigate the effect of multiplexed expression of gRNAs flanking a short stretch of DNA on reversion and mutation frequencies using yEvolvR. Phenotypic assays demonstrate that higher reversion frequencies are observed when expressing multiple gRNAs simultaneously. Next generation sequencing reveals a synergistic effect of multiple gRNAs on mutation frequencies, which is more pronounced in a mutant with a partially defective DNA mismatch repair system. Additionally, we characterize a galactose-inducible yEvolvR, which enables temporal control of mutagenesis. This study demonstrates that multiplex expression of gRNAs and induction of mutagenesis greatly improves the capabilities of yEvolvR for generation of genetic libraries in vivo.

CRISPR-Associated Transposase for Targeted Mutagenesis in Diverse Proteobacteria.

Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria (Burkholderia thailandensis and Pseudomonas putida, respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium Agrobacterium fabrum. In B. thailandensis, we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST's utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.

WAX INDUCER 1 Regulates ß-Diketone Biosynthesis by Mediating Expression of the Cer-cqu Gene Cluster in Barley.

Plant surface properties are crucial determinants of resilience to abiotic and biotic stresses. The outer layer of the plant cuticle consists of chemically diverse epicuticular waxes. The WAX INDUCER1/SHINE subfamily of APETALA2/ETHYLENE RESPONSIVE FACTORS regulates cuticle properties in plants. In this study, four barley genes homologous to the Arabidopsis thaliana AtWIN1 gene were mutated using RNA-guided Cas9 endonuclease. Mutations in one of them, the HvWIN1 gene, caused a recessive glossy sheath phenotype associated with ß-diketone deficiency. A complementation test for win1 knockout (KO) and cer-x mutants showed that Cer-X and WIN1 are allelic variants of the same genomic locus. A comparison of the transcriptome from leaf sheaths of win1 KO and wild-type plants revealed a specific and strong downregulation of a large gene cluster residing at the previously known Cer-cqu locus. Our findings allowed us to postulate that the WIN1 transcription factor in barley is a master mediator of the ß-diketone biosynthesis pathway acting through developmental stage- and organ-specific transactivation of the Cer-cqu gene cluster.

Efficient traceless modification of the P1 bacteriophage genome through homologous recombination with enrichment in double recombinants: A new perspective on the functional annotation of uncharacterized phage genes.

The advent of high-throughput omic technologies has caused unprecedented progress in research on bacteriophages, the most abundant and still the least explored entities on earth. Despite the growing number of phage genomes sequenced and the rejuvenation of interest in phage therapy, the progress in the functional analysis of phage genes is slow. Simple and efficient techniques of phage genome targeted mutagenesis that would allow one to knock out particular genes precisely without polar effects in order to study the effect of these knock-outs on phage functions are lacking. Even in the case of model phages, the functions of approximately half of their genes are unknown. P1 is an enterobacterial temperate myophage of clinical significance, which lysogenizes cells as a plasmid. It has a long history of studies, serves as a model in basic research, is a gene transfer vector, and is a source of genetic tools. Its gene products have structural homologs in several other phages. In this perspective article, we describe a simple and efficient procedure of traceless P1 genome modification that could also serve to acquire targeted mutations in the genomes of certain other temperate phages and speed up functional annotations of phage genes.

Genome editing in rice mediated by miniature size Cas nuclease SpCas12f.

Cas9 derived from Streptococcus pyogenes (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues). Recently, targeted mutagenesis in human cells and maize using Cas12f derived from Syntrophomonas palmitatica (SpCas12f)-a very small Cas of 497 a.a, which is a more suitable size for virus vectors-was reported. However, there are no reports of genome editing using SpCas12f in crops other than maize. In this study, we applied SpCas12f to genome editing in rice-one of the most important staple crops in the world. An expression vector encoding rice codon-optimized SpCas12f and sgRNA for OsTubulin as a target was introduced into rice calli by Agrobacterium-mediated transformation. Molecular analysis of SpCas12f-transformed calli showed that mutations were introduced successfully into the target region. Detailed analysis by amplicon sequencing revealed estimated mutation frequencies (a ratio of the number of mutated calli to that of SpCas12f-transformed calli) of 28.8% and 55.6% in two targets. Most mutation patterns were deletions, but base substitutions and insertions were also confirmed at low frequency. Moreover, off-target mutations by SpCas12f were not found. Furthermore, mutant plants were regenerated successfully from the mutated calli. It was confirmed that the mutations in the regenerated plants were inherited to the next-generation. In the previous report in maize, mutations were introduced by treatment with heat shock at 45°C for 4 h per day for 3 days; no mutations were introduced under normal growth conditions at 28°C. Surprisingly, however, mutations can be introduced without heat-shock treatment in rice. This might be due to the culture conditions, with relatively higher temperature (30°C or higher) and constant light during callus proliferation. Taken together, we demonstrated that SpCas12f can be used to achieve targeted mutagenesis in rice. SpCas12f is thus a useful tool for genome editing in rice and is suitable for virus vector-mediated genome editing due to its very small size.

Ribonucleoprotein (RNP)-Mediated Targeted Mutagenesis in Barley (Hordeum vulgare L.).

The crop species barley is a genetic model for the small grain temperate cereals. Thanks to the availability of whole genome sequence and the development of customizable endonucleases, site-directed genome modification has recently revolutionized genetic engineering. Several platforms have been established in plants, with the most flexible one offered by the clustered regularly interspaced short palindromic repeats (CRISPR) technology. In this protocol, commercially available synthetic guide RNAs (gRNAs), Cas enzymes, or custom-generated reagents are used for targeted mutagenesis in barley. The protocol has been successfully used with immature embryo explants to generate site-specific mutations in regenerants. As the double-strand break-inducing reagents are customizable and can be efficiently delivered, pre-assembled ribonucleoprotein (RNP) complexes allow efficient generation of genome-modified plants.

Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas Systems.

CRISPR/Cas has been established for targeted mutagenesis in many plant species since 2013, including Brassica napus and Brassica oleracea. Since that time, improvements have been made in terms of efficiency and choice of CRISPR systems. This protocol encompasses improved Cas9 efficiency and an alternative Cas12a system, allowing more challenging and diverse editing outcomes to be achieved.

Genome Editing of Medaka.

Medaka (Oryzias latipes), along with zebrafish (Danio rerio), is a useful experimental model fish. Here, we describe a simple method for generating medaka gene knockout strains using an automated microchip electrophoresis system. We also describe a method for targeted gene knockin using a plasmid carrying a sequence that does not cause off-target effects in medaka. Additionally, knockin method without plasmid cloning is described.

De novo PAM generation to reach initially inaccessible target sites for base editing.

Base editing by CRISPR crucially depends on the presence of a protospacer adjacent motif (PAM) at the correct distance from the editing site. Here, we present and validate an efficient one-shot approach termed 'inception' that expands the editing range. This is achieved by sequential, combinatorial base editing: de novo generated synonymous, non-synonymous or intronic PAM sites facilitate subsequent base editing at nucleotide positions that were initially inaccessible, further opening the targeting range of highly precise editing approaches. We demonstrate the applicability of the inception concept in medaka (Oryzias latipes) in three settings: loss of function, by introducing a pre-termination STOP codon in the open reading frame of oca2; locally confined multi-codon changes to generate allelic variants with different phenotypic severity in kcnh6a; and the removal of a splice acceptor site by targeting intronic sequences of rx3. Using sequentially acting base editors in the described combinatorial approach expands the number of accessible target sites by 65% on average. This allows the use of well-established tools with NGG PAM recognition for the establishment of thus far unreachable disease models, for hypomorphic allele studies and for efficient targeted mechanistic investigations in a precise and predictable manner.

Targeted Mutagenesis in Mice Using a Base Editor.

Base editors, such as cytosine and adenine base editors, are composed of nickase Cas9 (nCas9) and deaminase and serve as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based enzymatic tools for specific nucleotide substitutions. They are mainly the most effective genome editing tools for introducing point mutations, such as C-to-T and A-to-G conversions. The enhanced base editor, a C-to-G base editor (CGBE), can perform other nucleotide substitutions, such as C-to-G conversions. Here, we introduce a method for generating mouse models with point mutations using a base editing system.

Peculiar properties of tuber starch in a potato mutant lacking the α-glucan water dikinase 1 gene GWD1 created by targeted mutagenesis using the CRISPR/dMac3-Cas9 system.

Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze-thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.

Evaluation of genome and base editing tools in maize protoplasts.

Introduction: Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful. Methods: To this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established. Results and discussion: Mutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing.