What are the multiplex CRISPR strategies?

The two main multiplex CRISPR strategies are: multi-cassette (monointrinsic) strategies and single-cassette strategies(polyintrinsic). 

1. In multi-cassette strategies, each gRNA functions independently with its own set of control elements—a promoter to start transcription and a terminator to end it. Researchers typically need to clone multiple gRNA sequences individually to assess their activity. To achieve multiplexing in a CRISPR experiment, one approach is to insert multiple individually cloned gRNA plasmids into the cell line. This method works efficiently in easily transfected cell lines because each gRNA plasmid has a higher probability of reaching the same cell. Alternatively, researchers can use multiple gRNA expression cassettes into a single vector.
2. In the use of polycistronic cassettes, multiple gRNAs are produced from a single promoter, providing a resolution for the challenges encountered with monocistronic gRNA expression. Among these solutions, the polycistronic tRNA-gRNA (PTG) system is significant. In this system, gRNAs are integrated with transfer RNA (tRNA) genes. During transcription, the tRNA-gRNA transcript undergoes processing by tRNases to generate active gRNAs.