One-step assembly of large CRISPR arrays enables multi-functional targeting and reveals constraints on array design

Abstract

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.

TheFrancisella novicidaCas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays

“…The clearance assays were conducted by transforming a plasmid encoding a CRISPR array into an E. coli strain harboring an FnCas12a-expressing plasmid and a separate plasmid encoding the target sequence and canonical PAM (Figure 1(a)). Clearance activity could then be assessed based on the relative number of transformants with the tested array versus a no-spacer control (pC) [13,19]. The expectation was that Cas12a-mediated clearance of the target plasmid would sensitize cells to one of the antibiotics, greatly reducing the number of colonies compared to the no-spacer control.…”

“…We next investigated the ability of the two targeting constructs to direct DNA cleavage by FnCas12a in an all-E. coli cell-free TXTL system [21]. We previously used this system to interrogate the activity of CRISPR nucleases and guide RNAs, and it provides a quantitative and dynamic readout of nuclease activity [13,22]. As part of the assay, the FnCas12a plasmid and a CRISPR array plasmid were incubated in the TXTL mix for two hours.…”

“…In contrast, we found that DNA cleavage activity could be restored by replacing the 3ʹ repeat with the native terminal repeat from the endogenous CRISPR array, which contained mutations predicted to disrupt Cas12a binding and processing. Given the prevalence of disruptive mutations in native Cas12a terminal repeats [13], these repeats should be available for most Cas12a nucleases and may be interchangeable between different Cas12a nucleases.…”

“…However, eliminating the 3ʹ repeat enhanced the inhibitory effect for both tested spacers, whether in the single-spacer arrays (pT-1, pT-2) or as the downstream spacer in the two-spacer arrays (pT-12, pT-21) ( Figure 5). Another possibility is introducing the terminal repeat in the native CRISPR-Cas12a array from Francisella novicida [4,17], as these repeats harbor mutations predicted to disrupt processing and could help shield native arrays from any flanking secondary structures [13]. We found that using this repeat in arrays with the adjacent terminator restored the activity of the spacer in the single-spacer arrays (pR M T-1, pR M T-2) and the downstream spacer in the two-spacer arrays (pR M T-12, pR M T-21).…”

“…More recent examples of multiplexing applications with Cas12a include multi-site genome editing and gene activation [9,10], and opportunities are emerging for multiplexed base editing and DNA sensing using Cas12a [11,12]. Furthermore, recent advances have made the generation of large CRISPR arrays more straightforward and efficient [13]. With these opportunities and advances, the ensuing question is how to efficiently design CRISPR arrays to maximize the DNA targeting of Cas12a directed by each encoded guide RNA.…”