Cut-and-Paste DNA Insertion with Engineered Type V-K CRISPR-associated Transposases

CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. Type V-K CASTs offer distinct technological advantages over type I CASTs given their smaller coding size, fewer components, and unidirectional insertions. However, the utility of type V-K CASTs is hindered by a replicative transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid co-integrate products. Here, we overcome this limitation by engineering new CASTs with dramatically improved product purity. To do so, we compensate for the absence of the TnsA subunit in multiple type V-K CASTs by engineering a Homing Endonuclease-assisted Large-sequence Integrating CAST compleX, or HELIX system. HELIX utilizes a nicking homing endonuclease (nHE) fused to TnsB to restore the 5-prime nicking capability needed for dual-nicking of the DNA donor. By leveraging distinct features of both type V-K and type I systems, HELIX enables cut-and-paste DNA insertion with up to 99.3% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. Furthermore, we demonstrate the versatility of this approach by generating HELIX systems for other CAST orthologs. We also establish the feasibility of creating a minimal, 3-component HELIX, simplifying the number of proteins that must be expressed. Together, HELIX streamlines and improves the application of CRISPR-based transposition technologies, eliminating barriers for efficient and specific RNA-guided DNA insertions.

Effect of Juglone against Pseudomonas syringae pv Actinidiae Planktonic Growth and Biofilm Formation

Pseudomonas syringaepv Actinidiae (P. syringae) is a common pathogen causing plant diseases. Limoli proved that its strong pathogenicity is closely related to biofilm state. As a natural bacteriostatic agent with broad-spectrum bactericidal properties, juglone can be used as a substitute for synthetic bacteriostatic agents. To explore the antibacterial mechanism, this study was carried out to examine the inhibitory effect of juglone on cell membrane destruction, abnormal oxidative stress, DNA insertion and biofilm prevention of P. syringae. Results showed that juglone at 20 μg/mL can act against planktogenic P. syringae (107 CFU/mL). Specially, the application of juglone significantly damaged the permeability and integrity of the cell membrane of P. syringae. Additionally, juglone caused abnormal intracellular oxidative stress, and also embedded in genomic DNA, which affected the normal function of the DNA of P. syringae. In addition, environmental scanning electron microscope (ESEM) and other methods showed that juglone effectively restricted the production of extracellular polymers, and then affected the formation of the cell membrane. This study provided a possibility for the development and utilization of natural juglone in plants, especially P. syringae.

Genotyping Arabidopsis T-DNA lines v1

This protocol is used for genotyping Arabidopsis seedlings to test for the presence of a transfer DNA (T-DNA) insertion. By using two primer sets it is possible to determine whether a seedling is homozygous, heterozygous or azygous for an insertion in the predicted genomic location. To identify lines with T-DNA insertions in a gene of interest, you need the Arabidopsis Genome Identifier (AGI) number corresponding to the genomic locus (e.g. RCS1A = AT1G67090), then visit the Salk Institute T-DNA Express site to find all the mapped insertions at your locus of interest. Genotyping primers have been pre-designed for each T-DNA line, these can be retrieved from the Salk Institute T-DNA primer site, and ordered at any supplier of DNA oligonucleotides before starting the protocol. In the US T-DNA lines can be purchased from the Arabidopsis Biological Resource Center (ABRC) and in the UK and EU from the European Arabidopsis Stock Center (NASC). Recommended reading http://signal.salk.edu/tdnaprimers.2.html Setting up the PCR reaction Genotyping is performed with the Phire Direct PCR Mix, this includes the polymerase, nucleotides and salts necessary for amplification. We use the “dilution protocol” which involves taking a small leaf disk and homogenizing it in dilution buffer using a gel tip (see manufacturer’s instructions for more details.)

An Adenylate Kinase OsAK3 Involves Brassinosteroid Signaling and Grain Length in Rice (Oryza sativa L.)

Background Grain size is one of the major determinants of cereal crop yield. As a class of plant polyhydroxysteroids, brassinosteroids (BRs) play essential roles in the regulation of grain size and plant architecture in rice. In a previous research, we cloned qGL3/OsPPKL1 encoding a protein phosphatase with Kelch-like repeat domains, which negatively regulates BR signaling and grain length in rice. Results Here, we screened qGL3-interacting proteins (GIPs) via yeast two-hybrid assay and analyzed the phenotypes of the T-DNA insertion mutants of GIPs. Among these mutants, mutant osak3 presents shorter grain length and dwarfing phenotype. OsAK3 encodes an adenylate kinase, which regulates grain size by controlling cell expansion of rice spikelet glume. Overexpression of OsAK3 resulted in longer grain length. OsAK3 interacts with qGL3 in vivo and in vitro. Lamina inclination, coleoptile elongation and root inhibition experiments showed that the osak3 mutant was less sensitive to exogenous brassinolide (BL) treatment. The transcriptional level of OsAK3 was up-regulated under BL induction. In addition, RNA-Seq data indicate that OsAK3 is involved in a variety of biological processes that regulate BR signaling and grain development in rice. Conclusions Our study reveals a novel BR signaling component OsAK3 in the regulation of grain length, and provides novel clues for uncovering the potential functions of OsAK3 in rice growth and development.

Genotyping Arabidopsis T-DNA lines v3

This protocol is used for genotyping Arabidopsis seedlings to test for the presence of a transfer DNA (T-DNA) insertion. By using two primer sets it is possible to determine whether a seedling is homozygous, heterozygous or azygous for an insertion in the predicted genomic location. To identify lines with T-DNA insertions in a gene of interest, you need the Arabidopsis Genome Identifier (AGI) number corresponding to the genomic locus (e.g. RCS1A = AT1G67090), then visit the Salk Institute T-DNA Express site to find all the mapped insertions at your locus of interest. Genotyping primers have been pre-designed for each T-DNA line, these can be retrieved from the Salk Institute T-DNA primer site, and ordered at any supplier of DNA oligonucleotides before starting the protocol. In the US T-DNA lines can be purchased from the Arabidopsis Biological Resource Center (ABRC) and in the UK and EU from the European Arabidopsis Stock Center (NASC). Recommended reading http://signal.salk.edu/tdnaprimers.2.html Setting up the PCR reaction Genotyping is performed with the Phire Plant Direct PCR Mix, this includes the polymerase, nucleotides and salts necessary for amplification. We use the “dilution protocol” which involves taking a small leaf disk and homogenizing it in dilution buffer using a gel tip (see manufacturer’s instructions for more details.)

Predicting efficiency of writing short sequences into the genome using prime editing

Any short sequence can be precisely written into a selected genomic target using prime editing. This ability facilitates protein tagging, correction of pathogenic deletions, and many other exciting applications. However, it remains unclear what types of sequences prime editors can efficiently insert, and how to choose optimal reagents for a desired outcome. To characterize features that influence insertion efficiency, we designed a library of 2,666 sequences up to 69 nt in length and measured the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems. We discover that insertion sequence length, nucleotide composition and secondary structure all affect insertion rates, and that mismatch repair proficiency is a strong determinant for the shortest insertions. Combining the sequence and repair features into a machine learning model, we can predict insertion frequency for new sequences with R = 0.69. The tools we provide allow users to choose optimal constructs for DNA insertion using prime editing.

Effects of AtSPS on the Growth and Development of Arabidopsis thaliana under Abiotic Stress

Aims: SPS (Sucrose phosphate synthase) participates in plant growth and yield formation, and plays an important role in plant stress resistance. This study used T-DNA insertion mutant of AtSPS in Arabidopsis as test material. The growth indexes and soluble sugar contents of Arabidopsis thaliana under salt stress, osmotic stress and low temperature stress were determined, which laid the foundation for further understanding the mechanism of SPS in plant growth and development and abiotic stress resistance. Study Design: In order to analyze the mechanism of SPS in plant growth and development and abiotic stress resistance, this study used T-DNA insertion mutant of AtSPS in Arabidopsis as test material. The growth indexes and soluble sugar contents of Arabidopsis thaliana under salt stress, osmotic stress and low temperature stress were determined. Place and Duration of Study: College of Biological Science and Technology, between December 2020 and May 2021. Methodology: The contents of soluble sugar in tomato fruits were measured with HPLC (High performance liquid chromatography). The growth indexes were determined. Results: The results showed that AtSPS played positive regulation roles in seed germination and seedling growth of Arabidopsis thaliana. However, under abiotic stress conditions, AtSPS mutant increased the contents of soluble sugar, suggesting that Arabidopsis thaliana seedlings might improve resistance through osmotic regulating substances. Conclusion: AtSPS played positive regulation roles in seed germination and seedling growth of Arabidopsis. Meanwhile, AtSPS mutant increased the contents of soluble sugar to increase resistance of Arabidopsis under abiotic stresses, and the growth and development were blocked, suggesting that SPS was negative regulatory element to resist abiotic stress.

A dual prokaryotic (E. coli) expression system (pdMAX)

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (β-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.

Constitutive expression of JASMONATE RESISTANT 1 elevates content of several jasmonates and primes Arabidopsis thaliana to better withstand drought

Jasmonates have a well-documented role in balancing the trade-off between plant growth and defense against biotic stresses. However, the role of jasmonate signaling under abiotic stress is less well studied. Here, we investigated the function of JASMONATE RESISTANT 1 (JAR1) in drought stress in Arabidopsis thaliana. JAR1 converts jasmonic acid (JA) to jasmonyl-L-isoleucine (JA-Ile), the major bioactive form of jasmonates. Comparison of a newly generated over-expression line (JAR1-OE) with jar1-11, a T-DNA insertion line in the JAR1 locus, and Col-0 revealed that constitutively increased JA-Ile production results in stunted growth and a delay in flowering. Upon water limitation, JAR1-OE plants retained more water in their leaves, showed reduced wilting and recovered better from drought stress than the wild type. By contrast, jar1-11 mutant plants were hypersensitive to drought. RNA-seq analysis and hormonal profiling of plants under control and drought stress conditions provided insight into the molecular reprogramming caused by the alteration in JA-Ile content. Especially JAR1-OE plants were affected in many adaptive systems related to drought stress, including stomatal density, stomatal aperture or the formation of reactive oxygen species (ROS). Overall, our data suggest that constitutively increased expression of JAR1 can prime Arabidopsis towards improved drought tolerance.