SUMMARYCRISPR-Cas systems inherently multiplex through their CRISPR arrays--whether to confer immunity against multiple invaders or by mediating multi-target editing, regulation, imaging, and sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report an efficient, one-step scheme called CRATES to construct large CRISPR arrays through defined assembly junctions within the trimmed portion of array spacers. We show that the constructed arrays function with the single-effector nucleases Cas9, Cas12a, and Cas13a for multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. We also applied CRATES to assemble composite arrays utilized by multiple Cas nucleases, where these arrays enhanced DNA targeting specificity by blocking off-target sites. Finally, array characterization revealed context-dependent loss of spacer activity and processing of unintended guide RNAs derived from Cas12a terminal repeats. CRATES thus can facilitate diverse applications requiring CRISPR multiplexing and help elucidate critical factors influencing array function.

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Abstract. In this paper we introduce Self-Organizing Map-based techniques that can reveal structural cluster changes in two related data sets from different time periods in a way that can explain the new result in relation to the previous one. These techniques are demonstrated using a real-world data set from the World Development Indicators database maintained by the World Bank. The results verify that the methods are capable of revealing changes in cluster strucure and membership, corresponding to known changes in economic fortunes of countries.