Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases forin vivoGenome Editing

Abstract

1CRISPR-based genome editing is an enabling technology with potential to dramatically transform multiple industries. Identification of additional editing tools will be imperative for broad adoption and application of this technology. A novel Type V, Class 2 CRISPR nuclease system was identified from Microgenomates and Smithella bacterial species (CRISPR from Microgenomates and Smithella, Cms1). This system was shown to efficiently generate indel mutations in the major crop plant rice (Oryza sativa). Cms1 are distinct from other Type V nucleases, are smaller than most other CRISPR nucleases, do not require a tracrRNA, and have an AT-rich protospacer-adjacent motif site requirement. A total of four novel Cms1 nucleases across multiple bacterial species were shown to be functional in a eukaryotic system. This is a major expansion of the Type V CRISPR effector protein toolbox and increases the diversity of options available to researchers.

A modular cloning toolkit for genome editing in plants

“…pFH18: The monocot optimised SmCms1 gene was amplified in fragments from the vector SmCms1 (kindly provided by Benson Hill, USA) (Begemann et al 2017) using primers FH70/FH71 and FH72/FH73 and the fragments were subcloned into pCR-BluntII-Topo. The SmCms1 gene was then assembled into pICH41308 (Weber et al 2011) (Addgene ID #47998) using a GG cut-ligation reaction via BpiI using the subcloned fragments and the annealed primer pairs FH68/FH69 and FH74/FH75.…”

“…Benson Hill (USA) is the source of the SmCms1 vector/sequence and the license to the licensed patent rights. pFH19: The monocot optimised MiCms1 gene was synthesised in two parts with BpiI overhangs by Twist Bioscience (San Francisco, USA) based on the published sequence (Begemann et al 2017). The two parts were assembled into pICH41308 (Weber et al 2011) (Addgene ID #47998) using a GG cut-ligation reaction via BpiI.…”

“…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.…”

“…The fragments were subcloned into pCR-BluntII-Topo using the Zero Blunt™ TOPO™ PCR Cloning Kit (ThermoFisher Scientific) and then assembled into pICH41308 [2] (Addgene ID #47998) using a GG cut-ligation reaction via BpiI. pFH18: The monocot optimised SmCms1 gene was amplified in fragments from the vector SmCms1 (kindly provided by Benson Hill, USA) [10] using primers FH70/FH71 and FH72/FH73 and the fragments were subcloned into pCR-BluntII-Topo. The SmCms1 gene was then assembled into pICH41308 [2] (Addgene ID #47998) using a GG cut-ligation reaction via BpiI using the subcloned fragments and the annealed primer pairs FH68/FH69 and FH74/FH75.…”

A modular cloning toolkit for genome editing in plants

“…Lachnospiraceae bacterium (LbCas12a; pFH17 and pFH47) as well as with four related Cms1 nucleases (pFH18-21) (Additional file 2: Table S5). LbCas12a [20], FnCas12a [21] and Cms1 [22] have all been shown to work in plants.…”