The Strand - Synthetic Biology Newsletter, Issue 12
||The evolution of gene engineering tools
Whole-genome sequencing is becoming faster and more affordable than ever...read more
||Available now—the next CRISPR-based genome editing tool, GeneArt® CRISPR Nuclease...read more|
|Tips & Tricks
Top 5 GeneArt® Strings™ cloning tricks...read more
Read about how our customers are using GeneArt® Strings™ DNA Fragments in their research...read more
CRISPR technology: top 10 questions
| Upcoming Events and Synthetic Biology Resources
Did you know?
You can use optimized synthetic genes for efficient RNAi rescue.
Gene optimization takes advantage of degeneracy of the genetic code, enabling expression of the same protein sequence by two or more different DNA sequences. This can be utilized for RNAi rescue experiments, through expression of an optimized DNA sequence where the complementary sites for the interfering RNAs are exchanged without altering the protein sequence. Upon co-transfection of the optimized gene with the interfering RNAs, you can assess the efficiency of phenotype rescue.
Learn more about our
Check out the top 10 ways to help ensure valid RNAi data
Hot from Social
Have you already met the two new bases, X and Y?
Scientists at Scripps Research Institute have created two new bases: d5SICS and dNaM, also known as X, and Y! Both were successfully inserted into E. coli and replicated by the endogenous replication machinery like natural DNA.
Read the original article
The evolution of gene engineering tools
|Whole-genome sequencing is becoming faster and more affordable than ever. Translating this vast amount of genomic information into stories that have functional phenotypic relevance at cellular and systemic levels is the goal of much of genomics today. Understanding how life works, developing genome-based clinical applications to diagnose and treat disease, and delivering on the promise of personalized medicine all rely on developing a detailed understanding of how genotype relates to the phenotypes of health and disease. Beginning nearly a century ago, researchers studying the genetics of inheritance sought to understand the relationship between genotype and phenotype by selectively modifying the genome and measuring how those changes affected phenotypes. Hermann Muller first used X-rays to induce mutations in Drosophila in the 1920s, and although many gene manipulation techniques have been introduced and refined over the decades, one of the principal desires of genome researchers has been to precisely target genetic modifications. That targeting accuracy is the promise of modern genome editing.
Genome editing—precise, site-specific DNA modification—can now be achieved through the use of chimeric protein constructs that consist of a sequence-specific binding protein linked to a non-specific endonuclease that cleaves DNA a predictable distance from the binding site. The DNA-binding domains of both transcription activator–like (TAL) effectors and zinc-finger proteins are known and programmable, and that knowledge can be used to create customized proteins that bind specifically to virtually any desired DNA sequence. More recently, clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated (Cas) endonucleases, have also been used for genomic editing. Like the TAL effector nucleases (TALENs) and chimeric zinc-finger nucleases (ZFNs), these RNA-guided endonuclease (RGEN) systems also have modular DNA recognition and cleavage functions; —by engineering the DNA-recognition components, the endonuclease components of CRISPR/Cas systems can be targeted with high specificity to cut any genomic sequence desired.
Available now — the next CRISPR-based genome editing tool, GeneArt® CRISPR Nuclease mRNA
Instead of a large vector containing the required expression cassettes:
Quicker—ready for transfection, no cloning required
Top 5 GeneArt® Strings™ cloning tricks
GeneArt® Strings™ DNA Fragments can be used to quickly obtain your expression plasmid. Ordering via the GeneArt® online order portal is easy to use, includes our optimization function, and allows you to order DNA fragments up to 3,000 bp. Strings™ DNA fragments are typically produced in 5–8 business days and are suitable for any cloning strategy and an affordable alternative to ordering fully cloned gene synthesis.
Here are 5 tips for optimal design, cloning, and handling of Strings™ fragments:
- Always use the gene optimization function of the portal to improve your expression result. Even if Strings™ fragments are not ordered through the portal but are instead orderd using the Excel® form (can be downloaded from the webpage), it is recommended that you use the gene optimization function of the portal and then simply copy and paste the optimized sequence into the Excel® sheet for ordering. If a sequence is not producible as a Strings™ fragment, you will receive a portal message. In these cases, gene optimization can often be used to resolve the problems so that a Strings™ fragment can be produced.
- For optimal success of your desired downsteam cloning approach for the Strings™ DNA fragments, make sure to add appropriate 5’ and 3’ nucleotides to the ends of your sequence; e.g., restriction sites or overlaps for seamless cloning. Refer to the GeneArt® Strings™ DNA Fragments webpage for further assistance regarding best practices for the design of your fragments.
- After you receive the tubes, spin down the contents before opening, add the appropriate amount of water to the bottom of the tube, and let it incubate at room temperature for at least 1 hour or overnight. Resuspend the DNA carefully before use.
- We typically deliver more than the guaranteed 200 ng. However, to avoid running out of material, you can use the primer sequences given in the documentation to PCR- amplify your DNA fragment.
- Depending on the length of the Strings™ DNA fragments, we recommend the following screening to find a correct clone with high likelihood (>90%):
- For Strings™ fragments up to 1 kb, sequence 2–4 full-length clones
- For Strings™ fragments 1–2 kb, sequence 3–5 full-length clones
- For Strings™ fragments 2–3 kb, sequence 4–8 full-length clones
Full-length clones can be obtained by colony PCR of the single colonies after plating the transformed bacteria or by miniprep analysis.
Find more information on GeneArt® Strings™ DNA Fragments design, cloning, applications, and more
Have tips and tricks you use in your lab? Send us your tips
Read about how our customers are using GeneArt® Strings™ DNA Fragments in their research.
GeneArt® Strings™ DNA Fragments are custom-made, uncloned, double-stranded linear DNA fragments from 100 base pairs (bp) to 3,000 bp in length, assembled from synthetic oligonucleotides using the same process developed for high-quality GeneArt® gene synthesis.
Dr. Hans-Ulrich Schmoldt from BioNtech, Germany, used Strings™ fragments for a 15-position alanine scan (replacement of each amino acid with alanine) and stated:
”I just wanted to give you feedback regarding your tip with Strings synthesis for an alanine scan. I must say I am really enthusiastic. I cloned the 15 Strings directly without PCR amplification and then sent 6 clones each for sequencing. Nearly all clones were correct, which meant that I could finalize all cloning within 1.5 weeks.”
Dr. Justin B. Siegel, Assistant Professor at the UC Davis Genome Center and Department of Biochemistry & Molecular Medicine, used Strings™ fragments for synthesis of enzyme genes.
“DNA Strings have been a truly enabling technology for my lab. Before DNA Strings, my students spent a significant amount of time synthesizing genes from oligos, as this was significantly cheaper than ordering a standard gene. However, as an enzyme design lab, I want my students’ time primarily spent on the design and analysis of their engineered enzymes, not the construction. DNA Strings have provided us with a cost-effective way to readily obtain single genes to dozens of genes without needing to spend significant time on their construction.”
Published applications for Strings™ fragments:
In a recent publication, Scholey et al. used GeneArt® Strings™ DNA Fragments for mutation analysis of the kinesin-5 BASS domain, which plays a critical role in spindle formation during mitosis and is therefore considered a potential target for anti-cancer therapy.
In a second example, Hitachi et al. also used GeneArt® Strings™ DNA Fragments for mutagenesis. The authors investigated regulators of skeletal muscle mass and identified miR-486 microRNA as an important player.
Real-time PCR controls
Hornok et al. used a synthetic Strings™ fragment as a control for their real-time PCR experiments. Their research was aimed at investigating the distribution of Ixodes ricinus ticks in Hungary that carry human- pathogenic Candidatus Neoehrlichia mikurensis bacteria.
Optimized expression of coding sequences
Hartwig et al. identified and isolated a variant of patchoulol synthase (PTS) involved in plant terpenoid synthesis. They overcame expression difficulties of this plant sequence in bacteria by optimizing the sequence (using third-party software) and synthesizing the resulting sequence as GeneArt® Strings™ DNA Fragments.
Learn more about GeneArt® Strings™ DNA Fragments
Recently published your data? Share your data with us, and we’ll feature it in our newsletter.
Email us at SynBioNews@lifetech.com
CRISPR technology: top 10 questions
Q. What are the advantages of knockouts using TAL or CRISPR, compared to vector-based stable shRNA?
A. Because TAL and CRISPR edit the genome, modifications are typically achieved more efficiently. In the case of stable shRNA, the expressed shRNA works at the transcript level. Therefore, the effect of knockdown in gene expression depends on the level of expression of the shRNA, the activity of the promoter at the locus where the shRNA is stably integrated, and the ratio of shRNA to mRNA transcripts.
Q. Several articles have discussed the nonspecific activity of Cas9. Is it necessary to design more than one oligo for a gene?
A. Off-target effects can be minimized by carefully designing crRNA target oligos and avoiding homology with other regions in the genome.
Q. How can the changes be introduced into the genome?
A. By either oligo or transgene fragments. Point mutations can be a single base or amino acid mutation.
Q. Must the oligos be 5' phosphorylated?
A. Standard oligos would suffice. Try Life Technologies™ value oligos.
Q. If a target protein is very big and multifunctional, which is the best site to design guide RNA (gRNA) for? How do I check whether the introduced mutation blocks protein synthesis? How do I check whether the effect is due to target mutation?
A. The first few exons would be best (the closer to the promoter, the earlier the transcript termination). The gRNA efficiency depends on the accessibility of the locus as well as the chromatin structure at that location. It is advisable to design and test a few target sites, and you can always design gRNA to as many sites as you desire. Non–CRISPR-related mutations can be identified using a non–CRISPR-treated sample as a control followed by a GeneArt® Genomic Cleavage Detection assay. Standard western blot analysis should report the resulting protein levels.
Q. How do I design a guide RNA (gRNA) to insert a selection cassette (e.g., neomycinR)?
A. Using the gRNA oligo design strategy in the CRISPR manual, you can design guide RNA to the preferred locus for insertion of the neomycinR cassette. The cassette (neomycinR) itself can be inserted by leveraging homologous recombination, in which case the neomycinR cassette should contain locus-specific homology arms.
Q. Do you have CRISPR technology available for use in plants, i.e., a build-it-yourself plant-specific CRISPR cataloged kit?
A. We do not have a build-it-yourself plant-specific CRISPR cataloged kit yet, but we do have a custom vector design service. You can send us your vector design, and we can build a vector that includes a plant expression cassette and other necessary elements of your choice. Send your inquiry to CRISPR@lifetech.com, and we can assist you with your project design and quote.
Q. Does the requirement for a PAM-sequence motif on the genomic target inhibit targeting flexibility? What if there is no PAM sequence at the locus of interest?
A. While PAM is a necessary requirement, NGG (the PAM for the S. pyogenes–based CRISPR-Cas9 system) occurs quite often in a gene, so the chances are high that you will find a NGG PAM in your gene of interest. If it is absent, you could go with the GeneArt® Precision TAL effector–based nuclease.
Q. Is it possible to order Life Technologies™ custom plasmids with a different promoter (i.e., one that is species-specific or cell type–specific)? Will you offer a service to generate custom cell lines using CRISPR?
A. Yes; for all inquiries contact CRISPR@lifetech.com, and we can assist you with your project design and quote.
Q. What are the differences/benefits of the GeneArt® Genomic Cleavage Detection Kit?
A. The GeneArt® Genomic Cleavage Detection Kit supplies all key reagents to enable you to complete the entire cleavage efficiency assay workflow (starting from cell lysis all the way to % indel analysis). Other approaches (e.g., SURVEYOR® Mutation Detection Kit or CEL I assays) require the purchase of one or more components separately.
Have questions to ask us? Send us your questions