CRISPRi-based circuits to control gene expression in plants

Muhammad Adil Khan, Gabrielle Herring, Jia Yuan Zhu, Marina Oliva, Elliott Fourie, Benjamin Johnston, Zhining Zhang, Jarred Potter, Luke Pineda, Jahnvi Pflueger, Tessa Swain, Christian Pflueger, James P B Lloyd, David Secco, Ian Small, Brendan N Kidd, Ryan Lister

Research output: Contribution to journalArticlepeer-review

Abstract

The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts. We determine the optimal processing system to express single guide RNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. The performance of a NOR gate in stably transformed Arabidopsis plants demonstrates the system's programmability and reversibility in a complex multicellular organism. Furthermore, cross-species activity of CRISPRi-based logic gates is shown in Physcomitrium patens, Triticum aestivum and Brassica napus protoplasts. Layering multiple NOR gates together creates OR, NIMPLY and AND logic functions, highlighting the modularity of our system. Our CRISPRi circuits are orthogonal, compact, reversible, programmable and modular and provide a platform for sophisticated spatiotemporal control of gene expression in plants.

Original languageEnglish
Number of pages20
JournalNature Biotechnology
DOIs
Publication statusE-pub ahead of print - 20 May 2024

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