Editing genomes and regulating gene expression has become standard practice in many molecular biology labs.
This powerful technology has already begun to revolutionize several fields in biology facilitating new industrial, agricultural, and medical applications. Multiple enzymatic methods exist to edit genomes, including zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), transposases, and nucleases targeted to DNA by a small RNA derived from clustered regularly interspaced short palindromic repeats (CRISPR) in bacteria. Of these methods, CRISPR is the most versatile and commonly utilized.
Genome editing can be achieved by taking advantage of the double-stranded DNA breaks created by a targeted nuclease. These cleavage sites are most frequently repaired by the error-prone non-homologous end joining (NHEJ) repair pathway. NHEJ often results in inactivation or truncation of the targeted gene, a result that can help researchers test the role of a particular gene in their pathway of interest. In order to change the DNA sequence at the cleavage site to a desired sequence, a homologous ssDNA or dsDNA template is supplied that can support homology-directed repair (HDR). The HDR template DNA has ends which are homologous to the genomic DNA sequence on either side of the cleavage site and can mediate HDR of the lesion. This method can be used to introduce small to several kilobase sized inserts into the genome. In doing so, entire genetic circuits can be introduced into the genome that modify or enhance existing pathways or result in entirely novel outcomes such as production of an antigen that targets a cancer cell for T cell killing.
To accurately make edits in the genome using CRISPR-Cas9, it is of paramount importance that the targeting RNA and any HDR template are either error-free or of very high fidelity. Off-target cleavage by Cas9 can occur even with a perfect guide RNA sequence and occurs more frequently with a mixed population of guide RNA sequences. Additionally, the low frequency of HDR after DNA cleavage means that any delivered HDR template must be either error-free or very high fidelity in order to maximize the likelihood of success in editing the genome when HDR occurs. Arbor Biosciences provides high quality, affordable, genome editing solutions through error-free plasmid DNA for HDR and high fidelity sgRNA and long ssDNA HDR templates that allow effective editing and facilitate customization for any system of interest. Long ssDNA HDR templates have the advantage of typically outperforming plasmid-based HDR templates for repairing double-stranded DNA breaks. Our myCRISPR™ product lines are adaptable to a vast array of genome editing applications as they are derived from our myDNA™ error-free DNA synthesis pipeline. Additionally, our myLib® oligo libraries can be used to make guide RNA libraries that target thousands of different genomic sites. myLib® oligos can be delivered as dsDNA, ready for cloning into any plasmid system of choice.
While many researchers utilize CRISPR-Cas9 systems for genome editing, Arbor Biosciences can synthesize custom DNA for any desired genome editing technique. DNA synthesis can be a long and tedious process, so let our resident experts do the work instead.
DNA Synthesis Service
The myCRISPR™ product line is adaptable to a vast array of CRISPR-Cas9 related applications as they are derived from our myDNA™ error-free custom DNA synthesis pipeline. Custom DNA synthesis can be helpful at several stages of genome editing including building custom plasmids for delivery of sgRNA, making HDR templates for insertion of novel sequences, or templates for transcription of custom sgRNA sequences that can then be delivered in a ribonucleoprotein complex with Cas9 or a related endonuclease. To synthesize DNA constructs, we enrich for the highest quality oligos to be used as building blocks to construct your DNA sequence of choice. This avoids time-consuming error-correction procedures and allows Arbor Biosciences to offer fast turnaround times. Each DNA sequence delivered is put through a rigorous quality control process and is delivered fully sequenced using our next generation sequencing platform. Perform genome editing with confidence using our high quality DNA and RNA products.
Barrangou R et al. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science