Expanding the application of anti-CRISPR proteins in plants for tunable genome editing
Dear Editor, clustered regularly interspaced short palindromic repeats/CRISPR associated protein (CRISPR/Cas) systems have revolutionized genome engineering in plants via specific control of genetic modifications and transcriptional activities. However, knockout or overexpression of many critical genes may cause pleiotropic effects, which could be limited by conditional and tissue-specific gene modifications. Hence, it is necessary to develop spatially and temporally controllable CRISPR/Cas-based tools for precise genome engineering. Anti-CRISPR (Acr) proteins, natural inhibitors for CRISPR/Cas systems, have utility in biodesign strategies aimed at regulating Cas activities. Acr proteins inhibit CRISPR/Cas activities by either blocking (i) DNA-binding activity or (ii) DNA cleavage activity of Cas proteins. Multiple Acr proteins have been tested in mammalian cells and yeast (Saccharomyces cerevisiae), including AcrIIA4 (inhibits SpCas9), AcrVA1 (inhibits Cas12a), and AcrIIA5 (potentially inhibits all Cas9 orthologs) . Acr proteins can potentially regulate Cas activity at the post-translational level. For example, AcrIIA4 has been used to limit CRISPR/Cas activity to particular environments (e.g. blue-light) in nonplant cells. Also, cell-specific genome editing mediated by CRISPR/Cas9 was achieved through microRNA-dependent expression of Acr proteins in human cells. However, Acrs have not been widely used for tunable genome editing in plants. So far, only AcrIIA4 and AcrVA1 have been evaluated in a single plant species (Nicotiana benthamiana) based on transient expression through leaf infiltration and viral delivery. AcrIIA5 activity remains to be evaluated in plants, and performance differences between transient and stable expression of Acrs remain unanswered. Therefore, we evaluated the performance of AcrIIA4 and AcrIIA5 activities in herbaceous and woody plant species using both transient expression and stable transformation approaches. We tested the effects of AcrIIA4 and AcrIIA5 activities on the SpCas9-based adenine base editor (ABE7) in the herbaceous plants Arabidopsis (Arabidopsis thaliana) and N. benthamiana, and the woody plant hybrid poplar “717” (Populus tremula × P. alba hybrid clone INRA 717-1B4), using both leaf-infiltration and protoplast-based transient expression. The activity of AcrIIA4 on ABE7 was further investigated in Arabidopsis via Agrobacterium tumefaciens-mediated stable genetic transformation.
- Research Organization:
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Grant/Contract Number:
- AC05-00OR22725
- OSTI ID:
- 1972250
- Alternate ID(s):
- OSTI ID: 1958067; OSTI ID: 1959609
- Journal Information:
- Plant Physiology (Bethesda), Journal Name: Plant Physiology (Bethesda) Vol. 192 Journal Issue: 1; ISSN 0032-0889
- Publisher:
- Oxford University PressCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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