Difference between revisions of "Part:BBa K1689000"

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sgRNA generator

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system is originally from prokaryotic immune system [1], in which CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9 (Figure 1)[2]. However, the dual-tracrRNA:crRNA (Also called sgRNA), when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage[3].

The Part: sgRNA Generator was designed base on the type II CRISPR/Cas9 system (Figure 2) including T7 promotor, lacZa', crRNA(tracrRNA) and T7 terminator. The sequence of crRNA(tracrRNA) consists of 5’-terminal spacer-derived guide sequence and repeat-derived 3’-terminal sequence, but without the 17-20bp target-specific sequence. So we are able to carry out Golden Gate Cloning to insert the target-specific sequence between two BsaI cutting sites(With the lacZ coding sequence we can also identify whether the cloning is successful through white-blue plaque selection). and we can obtain the whole sgRNA (crRNA and tracrRNA) through cell-free transcription (Figure 2).The yield of RNA product is about 2500 ng/μl using HiScirbe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs).

Figure 1. Schematic of type II RNA-mediated CRISPR/Cas immune pathway. Cas9 is guided by a two-RNA structure formed by trans-activating tracrRNA and targeting crRNA to cleave site-specifically–targeted dsDNA. The mature crRNA consists of 5’-terminal spacer-derived guide sequence and repeat-derived 3’-terminal sequence.

Figure 2. Schematic of sgRNA Generator backbone, containing T7 promotor, lacZ a’, crRNA(tracrRNA) and T7 terminator, with two BsaI cutting sites flanking lacZa' coding sequence.

Figure 3. Flow chart of Goleden Gate Cloning to insert target sequence into sgRNA Generator. When screening out the target sequence (with Protospacer Adjacent Motif, PAM adjacent to it), we can easily generate oilgos for Golden Gate Cloning via [http://2015.igem.org/Team:Peking/Modeling#Modeling-OligoGenerator Oligo Generator] and then build sgRNA Generator for cell-free transcription later.

References

1. Rodolphe Barrangou, Christophe Fremaux, Hélène Deveau, et al. CRISPR provides acquired resistance against viruses. Science, 2007, 315: 1709-1712.

2. Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011;471:602–7.

3. Martin Jinek, Krzysztof Chylinski, Ines Fonfara, et al. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012, 337: 816-821.

Sequence and Features