CRISPR-mediated multiplexed genetic manipulation

The relative ease of using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)associated proteins (Cas-proteins) in genome editing has rapidly made this RNA-guided endonuclease system the method of choice in almost all current applications involving genetic manipulations, with Cas9 from S. pyogenes (SpCas9) being the most well characterized and broadly used [1]. Compared to the early generations of programmable DNA endonucleases such as the Zincfinger nuclease (ZFN) or Transcription Activator-Like Effector Nuclease (TALEN), the CRISPR-Cas9 system is time-, labor-, and cost-effective. Mediated by a small guide RNA (sgRNA or gRNA), Cas9 can specifically introduce double-strand DNA breaks at pre-selected genomic loci which (i) contain a target sequence (also known as a protospacer) complementary to the typically 20-nt guide sequence of the gRNA, and (ii) are followed by a protospacer sequence adjacent motif (PAM, 5’-NGG3’ for SpCas9). Furthermore, by co-delivery of Cas9 and multiple gRNAs, the CRISPR/Cas9 system enables the simultaneous modification of several loci/genes in the same cell [2]. Currently, most CRISPR-Cas9-based studies rely on plasmid-based or viral vector mediated expression of the Cas9 and gRNAs. The generation of plasmids expressing multiple gRNAs has been a time-consuming procedure involving multiple steps of cloning. To facilitate CRISPR-mediated multiplexed gene editing applications, we have opted for the Golden-Gate assembly method and developed a system for rapid construction of plasmids expressing up to 30 gRNAs within one week [3]. Golden-Gate assembly has been an attractive and cost-effective method for rapidly assembly of multiple DNA fragments in a single reaction. This method uses a special class of enzymes (type IIS restriction enzymes) which recognize asymmetric DNA sequences and induce staggered DNA-cleavage outside of their recognition sites. Three type IIS restriction enzymes have been favored for Golden-Gate assembly: BbsI (5’-GAAGAC(2/6); Editorial