CRISPR Guide RNA Cloning for Mammalian Systems

Abstract

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

“…Generation of multiplexing gRNA vectors containing more than one gRNA expression cassette has been reported with a number of different methods 21,22,23,24,25,26,27 . While some employ simultaneous or sequential cloning of gRNA pairs 22,34 , others depend on Golden Gate assembly and several sequential cloning steps 25,35 . A special approach has been used by Xie et al, by employing the endogenous cellular tRNA processing system to cut several gRNAs out of a single progenitor transcript 36 .…”

“…Several useful methods have been described to generate gRNA vectors containing more than one gRNA expression cassette 21,22,23,24,25,26,27 .…”

High-throughput tagging of endogenous loci for rapid characterization of protein function

“…Three gRNA libraries with different frames were synthesized as oligo pools from Agilent. Each gRNA library was then PCR amplified from the initial oligo pool and cloned into the modified pSB700 vector using Golden Gate assembly as previously described 31 .…”

“…To construct the gRNA expression plasmids, pSB700-blasto (Addgene #167904) was used for SpCas9-specific gRNA expression and a modified pSB700-vector containing a SaCas9 compatible gRNA scaffold with a zeocin-resistance gene was used for SaCas9-specific gRNA expression. Vectors containing gRNAs were cloned by Golden Gate using Esp3I as described previously 31 . pCAS plasmid was constructed from a previous dual-Cas9 plasmid (Addgene #107320) by replacing the 3xHA sequence with a P2A sequence using Gibson assembly.…”

“…Each gRNA library was then PCR amplified from the initial oligo pool and cloned into the modified pSB700 vector using Golden Gate assembly as previously described 31 .…”