One-Step Assembly of Class 2 CRISPR Arrays for Multiplexed Cleavage and Regulation

Clustered Regularly Interspaced short palindromic Repeats (CRISPR) systems have gained widespread attention because they can be harnessed for genome editing and gene regulation in different organisms. Most Class 2 CRISPR effector proteins discovered so far have the ability to process CRISPR arrays into individual guide RNAs. However, the conserved repeat elements in the CRISPR array prevent its chemical synthesis, limiting our ability to use these arrays for multiplexed genome editing and gene regulation. To overcome this barrier, we have developed a novel assembly scheme for the single-step, efficient generation of large CRISPR arrays. The resulting arrays guided multiplexed DNA cleavage and gene regulation in Escherichia coli and Saccharomyces cerevisiae by the FnCpf1 and AsCpf1 nucleases. The cloning scheme was also extended to the generation of CRISPR arrays utilized by Cas9 for multiplexed DNA cleavage in E. coli and by C2c2 for multiplexed targeting in TXTL. Moreover, CRISPR arrays co-utilized by Cpf1 and Cas9 were also assembled and proved to be functional in E. coli. We also noticed that some functional spacers showed reduced or negligible activity in the context of CRISPR arrays. This platform offer simplified multiplexing with Cpf1 and other Class 2 effector proteins across the myriad applications of CRISPR technologies.