CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with the CRISPR/Cas technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRISPR/Cas tools in biological systems is therefore very necessary. Here, they developed four real-time detection systems that can spontaneously indicate the presence of active CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing, and CRISPRa in plants. Using the fluorescence-based molecular biosensors, they demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing, and CRISPRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.
Different CRISPR/Cas-based genome engineering tools, such as base editors (BEs), prime editors (PEs), CRISPR activation (CRISPRa), and interference (CRISPRi), have been developed recently. However, the CRISPR/Cas technology poses potential biosecurity risks, increasing ethical and safety concerns. One major concern is that unwanted DNA changes or “off-target” events might accidentally arise from the continuous expression of active CRISPR systems. The unwanted CRISPR/Cas activities can cause an unpredictable phenotypic change in plants, which can negatively affect long-term environmental sustainability and food and animal feed safety, especially in perennial plants with long life-cycle and/or vegetative reproduction. Therefore, it is necessary to develop a system that can detect the presence of active CRISPR/Cas tools in biological systems. Different CRISPR/Cas technologies have been tested in various plant species. However, methods for real-time detection of CRISPR systems have not yet been reported in plants. Given that Streptococcus pyogenes Cas9 (SpCas9) is currently the most widely used genome editor in plants, they developed several fluorescence-based biosensors to detect in planta activities of SpCas9-based CRISPR/Cas9, base editors, prime editors, and CRISPRa. One advantage of their function-based biosensors is that they do not require any prior knowledge of the DNA sequence for the CRISPR systems, which is a prerequisite for many DNA based detection methods.
Detection of CRISPR tools for genome editing and gene regulation in plants. (a) BS1 for detection of the CRISPR/Cas9 nuclease. (b) Detection of CRISPR/Cas9 by BS1 through Arabidopsis protoplast transformation and tobacco leaf infiltration. (c) Statistical analysis of GFP-positive cells with and without the CRISPR/Cas9 in Arabidopsis. (d) BS2 for detection of the adenine base editor (ABE). (e) BS3 for detection of the prime editor. (f) Detection of an ABE by BS2 through protoplast transformation in Arabidopsis and poplar, and tobacco leaf infiltration. (g–i) Statistical analysis of GFP-positive cells with and without ABE in Arabidopsis, poplar 717-1B4, and poplar WV94. (j) Detection of an ABE by BS2 through stable transformation in Arabidopsis. (k) Detection of prime editor PPE2 by BS3 through Arabidopsis protoplast transformation. (l) Statistical analysis of GFP-positive cells with and without PPE2. (m) BS4 for detection of CRISPRa. (n) Detection of CRISPRa by BS4 through Arabidopsis protoplast transformation. (o) Statistical analysis of mCherry-positive cells with and without the CRISPR–Act3.0 activation system. Scale bar, 100 μm. All data are presented as the mean ± SE (n = 5 independent scopes).
Plant-Based Biosensors for Detecting CRISPR-Mediated Genome Engineering Guoliang Yuan, Md. Mahmudul Hassan, Tao Yao, Haiwei Lu, Michael Melesse Vergara, Jesse L. Labbé, Wellington Muchero, Changtian Pan, Jin-Gui Chen, Gerald A. Tuskan, Yiping Qi, Paul E. Abraham, and Xiaohan Yang ACS Synthetic Biology 2021 10 (12), 3600-3603 DOI: 10.1021/acssynbio.1c00455