Abstract
One key advantage of the CRISPR/Cas9 system in comparison with other gene editing approaches lies in its potential for multiplexing. Here, we describe an elaborate procedure that allows the assembly of multiple gRNA expression cassettes into a vector of choice within a single step, termed ASAP(Adaptable System for Assembly of multiplexed Plasmids)-cloning. We demonstrate the utility of ASAP-cloning for multiple CRISPR-mediated applications, including efficient multiplex gene editing, robust transcription activation and convenient analysis of Cas9 activity in the presence of multiple gRNAs.
Highlights
The possibility of multiplexing is one of the advantageous features of the CRISPR/Cas[9] system compared with other gene editing approaches
For each individual gRNA expression cassette (GEC), the “PCR-on-ligation” reaction is performed by ligating annealed oligonucleotides, encoding the protospacer complementary region of the gRNA, into the pX330 backbone according to the original Zhang lab protocol[10], except
In comparison to some earlier reports, which were based on distinct destination vectors and required the prior purchase of defined plasmids or plasmid libraries[4,5,6,7], ASAP-cloning allows the utilization of many commonly available vectors as destination backbones, thereby enabling a variety of applications
Summary
The possibility of multiplexing is one of the advantageous features of the CRISPR/Cas[9] system compared with other gene editing approaches (reviewed by Dominguez et al.[1]). Elaborate primer design and novel utilization of pairs of isocaudomers (type II restriction enzymes having slightly different recognition sites but producing identical overhangs) allows for ligation of the generated inserts into type II restriction endonuclease (TII-RE) sites in a large variety of common expression vectors, enabling utilization of this technique in a wide range of applications. We demonstrate this utility by constructing different multiplex CRISPR vectors for various downstream applications. This method is termed “ASAP-cloning” (Adaptable System for Assembly of multiplexed Plasmids)
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