Clustered regularly interspaced short palindromic repeat (CRISPR) elements in bacteria evolved as a defense against bacteriophages that infect bacteria.
CRISPR elements are encoded into the bacterial genome as an array of unique protospacer sequences that are each immediately followed by direct repeat sequences. The protospacer and direct repeat sequences are transcribed into RNA as an array which are then processed into individual crRNA sequences, each specific for a different DNA sequence. The protospacer sequences are specific to bacteriophage genomic DNA and the direct repeat sequence binds a trans-activating crRNA (tracrRNA). The tracrRNA then mediates binding to the RNA-guided nuclease (e.g. Cas9)(1). The protospacer sequence can then guide the nuclease to its cognate DNA target located upstream of a protospacer adjacent motif (PAM). The RNA-directed Cas9 endonuclease from Streptococcus pyogenes and related endonucleases from other bacterial species have been used in multiple applications including gene knock-out or knock-in, gene repression or activation, protein tagging, labeling of genomic DNA, CRISPR-mediated genome editing and regulation, and genetic screening. CRISPR-mediated genome editing and regulation is a rapidly expanding field that requires accuracy, flexibility and customization. We offer several products, each fully customizable, to take full advantage of existing and new CRISPR techniques to make small and large edits in genomic regions of interest.
CRISPR-Cas9 and related endonucleases are already of great benefit to synthetic biologists who desire to generate specific genome changes in their organism of interest. This technique can also be used to regulate gene expression in vivo or in vitro utilizing a Cas9 protein that lacks nuclease activity but can still bind DNA (dCas9). This nuclease can be combined with a specific sgRNA and then used to fine-tune genetic circuits and to better control circuits with increased complexity. Our cell-free myTXTL® system can also be used to test the efficacy of Cas9 cleavage on DNA in vitro (2), which can aid high-throughput discovery or evolution of Cas9-related enzymes into nucleases with new or improved properties.
To accurately make edits in the genome using CRISPR-Cas9, it is of paramount importance that the targeting RNA and any homology-directed repair (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 can be further increased with a mixed population of guide RNA sequences. Similarly, the low frequency of HDR after Cas9 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. Arbor Biosciences can provide high quality genome editing solutions through error-free plasmid DNA for HDR and high fidelity sgRNA and long ssDNA HDR templates that allow effective editing and facile customization for your system of interest. Long ssDNA HDR templates have the advantage that they typically outperform plasmid-based HDR templates for repairing double-stranded DNA breaks.
R. Marshall et al. (2017) Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system. ACS Synth Biol.
R. Barrangou et al. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science.