Transfection is commonly used to deliver all manner of nucleic acids into cells—everything from simple reporter vectors to inhibitory RNAs. Most recently, transfection has been used to alter cells’ endogenous nucleic acids via gene editing for applications such as gene repair and the creation of disease models. However, gene-delivery difficulties have been hindering the use of these new tools in relevant cellular models such as stem cells and primary cells.
Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) enable targeted cleavage of DNA at a specific locus, opening the door for endogenous repair mechanisms to add, replace or remove DNA. But the effectiveness of these tools is contingent upon efficient delivery, proper design, the intrinsic properties of the locus of interest and the painstaking downstream processes used for characterization and selection. Life Technologies™ has developed products to aid in design and delivery of both TALENs and CRISPRs. Here we outline strategies for optimizing performance and minimizing downstream workflows.
Transfection efficiency and protein expression using a CRISPR vector. The vector contained an OFP reporter gene and was transfected with Lipofectamine® 2000 or Lipofectamine® 3000 reagent into (A) U2OS and (B) HepG2 cell lines. Bar graphs show reporter gene expression; images show fluorescence of corresponding cells expressing OFP
Design considerations
TALENs
Genome editing uses engineered nucleases in conjunction with endogenous repair mechanisms to alter cellular DNA. TAL effector proteins, such as TALENs, consist of constant N- and C-terminal domains (containing translocation and nuclear localization/activation signals, respectively) flanking a central repeat domain. Each repeat is 34 to 35 amino acids in length, with two centrally located residues that dictate the repeat’s affinity for different nucleotide targets. The combination and order of various repeat types define the genomic target-site specificity of a particular TAL effector.
Deciphering this TAL effector “code” led, recently, to the engineering of designer proteins that can target various functionality to essentially any open region of the chromosomes of bacteria, yeast, plants, insects, zebrafish and mammals [1][2]. Traditionally, cloning and designing these sequences has been technically challenging because of improper annealing of the repeat domains. But simpler commercial options do exist. Life Technologies offers a GeneArt® web design tool, for instance, that can be used to design, optimize and construct custom GeneArt Precision TALs.
Your choice of targets is almost unlimited. The predictability with which GeneArt Precision TAL effectors bind to exact DNA sequences makes it possible to target any sequence in the genome. The choice of the effector domain then determines whether the Precision TAL effector edits, activates or represses the targeted gene.
After you have your custom enzyme, you can assess its efficiency using the GeneArt Genomic Cleavage Detection Assay, which leverages mismatch-detection endonucleases to detect insertions and deletions (indels) generated during cellular nonhomologous end-joining (NHEJ) repair.
CRISPR
The CRISPR-Cas system used in gene editing is a prokaryotic adaptive immune system that uses an RNA-guided DNA nuclease to silence viral nucleic acids [3]. In bacteria, CRISPR loci are comprised of a series of repeats separated by segments of exogenous DNA approximately 30 base pairs in length, called spacers. The repeat-spacer array is transcribed as a long precursor and processed to generate small crRNAs that specify the target sequences to be cleaved by the Cas9 nuclease.
Life Technologies has developed a commercial implementation of the CRISPR-Cas system. The linearized GeneArt CRISPR Nuclease vectors provide rapid and efficient cloning of a guide RNA (gRNA) oligo that mimics the natural crRNA-tracrRNA chimera for specific targeting of the Cas9 nuclease to a genomic locus. This short gRNA should be designed for a target sequence that is 19 to 20 nucleotides in length and adjacent to a 3’ NGG motif (called a proto-spacer-adjacent motif, or PAM). It is important that it contain no significant homology to other genes.
Because the gRNA supplies the specificity, changing the Cas9 target requires only a change in the gRNA sequence. Multiple gRNAs also can be cloned into one vector when numerous gene alterations of the host genomic DNA are desired.
The GeneArt CRISPR Nuclease Vector Kits enable expression of all the functional components of the CRISPR-Cas genome-editing mechanism and enrichment of the cell population. The vectors are available with two reporters: orange fluorescent protein (OFP) for flow cytometry-based sorting and CD4 for bead-based enrichment, both of which enable the selection and enrichment of the Cas9 and CRISPR RNA-expressing cell population.
Efficient delivery
The transfection protocol for Lipofectamine® 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines.
After you have your TALEN or CRISPR constructs, you need to deliver them to your cells. This is where lipid-based delivery reagents, like Lipofectamine® 3000, should be used, as improved transfection will boost cleavage and recombination efficiency.
Lipofectamine 3000 is a novel reagent developed to improve transfection efficiency in difficult-to-transfect cell lines. Using it, researchers can minimize painstaking downstream processes in their genetic-engineering protocols. The following steps will help you optimize transfection for your cell line:
• Test two doses: The Lipofectamine 3000 protocol suggests testing two doses of the transfection reagent. The rationale behind this suggestion is that every lab cultures different cell lines in a different manner. Even from person to person, there can be differences in the starting culture of cells used for transfection. Performing the protocol with the indicated two doses will guarantee you reach the transfection “sweet spot” for the reagent for the cell type you are using.
• Use OptiMEM® reduced serum media: Lipofectamine 3000 uses a two-tube protocol. It is important to add Lipofectamine 3000 to OptiMEM in one tube, add the DNA and P3000™ reagent to OptiMEM in another tube and to mix each tube thoroughly. Then, mix equal amounts from each tube and incubate for five minutes at room temperature to form the transfection complexes. The lipid-DNA complex can then be added directly to cells in complete culture media (that is, media containing sera, antibiotics, etc.).
• Control cell passage number: Most cell types should be used for transfection experiments between four and 25 passages post-thawing.
• Optimize cell density: Cells should be 70% to 90% confluent on the day of transfection. This can improve efficiency by 10% to 15%.
• Keep your cells healthy: Maintain healthy cells in log phase during subculturing and make sure to use the proper culture media.
Editing efficiency depends on the design and delivery of your nucleic acids. Using GeneArt Precision TAL or GeneArt CRISPR Nuclease Vectors with the improved transfection capabilities of Lipofectamine 3000 will allow you to concentrate on what really matters: your science.
References
[1] Moscou, MJ, Bogdanove, AJ, “A simple cipher governs DNA recognition by TAL effectors,” Science, 326:1501, 2009. [PubMed ID: 19933106]
[2] Boch, J, et al., “Breaking the code of DNA binding specificity of TAL-type III effectors,” Science, 326:1509-12, 2009. [PubMed ID: 19933107]
[3] Jinek, M, et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, 337:816-21, 2012. [PubMed ID: 22745249]
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