One-step assembly of large CRISPR arrays enables multi-functional targeting and reveals constraints on array design
One-step assembly of large CRISPR arrays enables multi-functional targeting and reveals constraints on array design
SUMMARY CRISPR-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.
Denny Steven R.、Leenay Ryan T.、Slotkowski Rebecca A.、Ttofali Fani、Keung Albert J.、Beisel Chase L.、Liao Chunyu、Cecil Taylor D.
Department of Chemical and Biomolecular Engineering North Carolina State UniversityDepartment of Chemical and Biomolecular Engineering North Carolina State UniversityDepartment of Chemical and Biomolecular Engineering North Carolina State UniversityDepartment of Chemical and Biomolecular Engineering North Carolina State UniversityDepartment of Chemical and Biomolecular Engineering North Carolina State UniversityDepartment of Chemical and Biomolecular Engineering North Carolina State University||Helmholtz Institute for RNA-based Infection Research Josef-Schneider-Str. 2 / D15, D-97080 W¨1rzburgDepartment of Chemical and Biomolecular Engineering North Carolina State University||Helmholtz Institute for RNA-based Infection Research Josef-Schneider-Str. 2 / D15, D-97080 W¨1rzburgDepartment of Chemical and Biomolecular Engineering North Carolina State University
生物科学研究方法、生物科学研究技术分子生物学生物工程学
Cas9Cpf1C2c2CRATESCRISPR RNADNA assemblygenome editinggene regulation
Denny Steven R.,Leenay Ryan T.,Slotkowski Rebecca A.,Ttofali Fani,Keung Albert J.,Beisel Chase L.,Liao Chunyu,Cecil Taylor D..One-step assembly of large CRISPR arrays enables multi-functional targeting and reveals constraints on array design[EB/OL].(2025-03-28)[2025-06-15].https://www.biorxiv.org/content/10.1101/312421.点此复制
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