Date: 2018-12-02 05:53
Due to limitations in delivery methods and plasmid vector capacities, simultaneous production of numerous gRNAs needs a suitable strategy. Multiplexing of sgRNAs allows to lead Cas9-proteins to several target sites using only one single construct.
A major obstacle is to enable precise processing of the transcripts. A number of possibilities is available to overcome this hurdle.
Transcript processing using tRNAs
tRNAs are highly conserved in all living organisms. The precisely cleavage of both tRNA precursors ends can be utilised for in vivo transcript processing. Polycistronic tRNA-sgRNA constructs allow separation of sgRNA within the nucleoplasm, providing exact 5′-ends of sgRNAs (Xie et al. 2015).
Ribozymes for self-processing systems
Ribozymes are able to process themself after transcription, which allows the construction of self-processing multiplex systems. Gao and Zhao (2014) showed that the inclusion of a Hammerhead-ribozyme upstream and HDV (Hepatitis Delta Virus RNA)-ribozyme downstream of sgRNA enabled a precise cut of 5′ and 3′ sgRNA ends.References
Xie, K., Minkenberg, B., & Yang, Y. (2015). Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proceedings of the National Academy of Sciences, 112(11): 3570-3575.
Gao, Y., & Zhao, Y. (2014). Self‐processing of ribozyme‐flanked RNAs into guide RNAs in vitro and in vivo for CRISPR‐mediated genome editing. Journal of integrative plant biology, 56(4): 343-349.