Comparison of Efficiency and Specificity of CRISPR-Associated (Cas) Nucleases in Plants: An Expanded Toolkit for Precision Genome Engineering

Abstract

Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPRassociated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called 'off-targets' by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content correlates with efficiency and that Cas9 from Staphylococcus aureus is comparatively most efficient at inducing mutations. We also demonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.

A modular cloning toolkit for genome editing in plants

“…The cloning toolkit is an addition to previously published GG modules [7,[9][10][11] and includes new sequence-specific CRISPR/Cas nucleases (codon-optimised for both monocots and dicots), Pol II and Pol III promoters, as well as guide RNA backbone modules. The latter enable insertion of the guide sequence by cloning in a pair of annealed complimentary oligos without a PCR amplification step involved.…”

“…The modular cloning kit presented in the study enables quick and facile assembly of DNA constructs for genome editing applications in plants and is an addition to previously published collections of compatible GG modules [7,[9][10][11]. The kit includes modules encoding a number of CRISPR/Cas nucleases (SaCas9, StCas9, LbCas12a etc.)…”

“…We believe the presented modular cloning kit will become a valuable addition to the range of already available GG modules [7,9,10] and expect that plant researchers, working with both monocots and dicots, will find the presented molecular tools useful for various genome editing applications. We also believe our study will contribute towards wider adoption of the GG modular cloning system by plant researchers and consequently facilitate exchange of standardised molecular cloning parts across the research community.…”

“…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 multi-expression unit constructs using standard genetic parts, such as promoters, terminators, coding sequences etc. The system has already been successfully used for genome editing applications in plants [3,4,[8][9][10] but lacks modules encoding many of the newest genome editing reagents. Here we report on an expanded GG cloning toolkit for genome editing applications in monocot and dicot plants.…”

A modular cloning toolkit for genome editing in plants

“…FnCas12a crRNA backbone AGAT (N) 23 AAAA (N) 23 reverse complement Note: The corresponding promoter modules contain already the correct transcriptional start nucleotide for expression of the guides (A in case of U3 promoters, G in case of U6 promoters) at their 3'-end to allow transcription of all guides independent of their first nucleotide. If your target sequence starts with the correct transcriptional start nucleotide already, consider to not add it to your primer sequence (example: for pFH85, you would just order two primers AGCA (N) [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and AAAC (N) 20-2 reverse complement ). This does not apply to the Cas12a/Cms1 crRNAs, as the target sequence is anyway at the 3'end of the crRNA sequence.…”