Abstract
BackgroundCRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.ResultsHere we present an expanded cloning toolkit that contains 103 modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.ConclusionsWe believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.
Highlights
CRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants
The cloning toolkit is an addition to previously published Golden Gate (GG) modules [7, 9,10,11] and includes, among many new elements, modules encoding FnCas12a, LbCas12a, Cms1 nucleases, C-G to T-A and A-T to G-C base editors, Cas9 variants with alternative protospacer adjacent motif (PAM) specificities (SaCas9, StCas9, ScCas9 etc), polymerase II (Pol II) and polymerase III (Pol III) promoters, as well as guide RNA backbone modules
With the tRNAsgRNA system proven to be efficient in both monocot [6, 12] and dicot (Arabidopsis) [13] plant species, our modules enabling a straightforward PCR
Summary
CRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs. As genome editing applications in plants often rely on delivering multiple expression units into plant cells, including a selectable marker, a CRISPR/Cas nucleaseencoding gene and one or more guide RNAs, it is important to be able to assemble DNA constructs encoding such expression units and rapidly. The modular cloning (MoClo) system based on the Golden Gate (GG) cloning method [7] is highly flexible and versatile, and provides a means for quick and facile assembly of multiexpression unit constructs using standard genetic parts, such as promoters, terminators, coding sequences etc. We believe the toolkit will become a valuable addition to already existing GG-based tools for plant genome editing and be widely used by plant researchers across the community
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