ISAAA News - June 24, 2020

Experts from the University of Chinese Academy of Sciences introduced a new technique called CRISPR simultaneous and wide-editing induced by a single system (SWISS), which allows multi-functional genome editing in plants. The detailed description of SWISS is published in Genome Biology.

In this new method, the RNA aptamers engineered in crRNA scaffold recruit their cognate binding proteins fused with cytidine deaminase and adenosine deaminase to Cas9 nickase target sites, leading to multiplexed base-editing. When paired sgRNAs are used, SWISS can cause insertions or deletions in addition to base editing. When this method was tested in rice, the mutants generated exhibited efficiencies of cytosine conversion of 25.5%, adenine conversion of 16.4%, indels of 52.7%, and simultaneous triple mutations of 7.3%.

Based on the tests, the SWISS system is a powerful tool for multi-functional genome editing in plants.

For more findings, read the open-access article at Genome Biology.

Figure: Multiple RNA scaffolds and binding protein orthologs mediate efficient C-to-T conversion. a The CRISPR scaffold RNA-programmed multiplex genome editing system based on nCas9 nuclease. Abbreviation: BP, binding protein. b Architecture of the pOsU3-esgRNA-2×MS2 construct with two MS2 hairpins at the 3′-end of the esgRNA. Abbreviation: SUP4 Term, transcription terminator for the S. cerevisiae SUP4 tRNA gene. c Architectures of PBEc1-c5. Abbreviations: XTEN, 16-aa linker; NLS, nuclear localization signal; CaMV, cauliflower mosaic virus; Term, terminator. d Comparison of C-to-T conversion using a BFP-to-GFP reporter system by PBE and the five PBEcs in rice protoplasts (n = 3). Values and error bars indicate means ± s.e.m. of three independent experiments. e Architectures of PBEc6-c8. Abbreviations: XTEN, a 16-aa linker; NLS, nuclear localization signal; CaMV, cauliflower mosaic virus; Term, terminator. f Schematic of the scRNAs with MS2, PP7, boxB, or com RNA hairpins in the tetraloop and stem loop2 or the 3′-end of the sgRNA and esgRNA. g Comparison of C-to-T conversion using the BFP-to-GFP reporter system induced by various scRNAs and their cognate PBEcs in rice protoplasts (n = 3). The f6 aptamer hairpin binds MCP specifically. Two PP7 hairpin variants were adopted. Values and error bars indicate means ± s.e.m. of three independent experiments. h Comparison of C-to-T editing frequencies of rice endogenous genes induced by four scRNAs and their cognate PBEcs (n = 3). An untreated protoplast sample served as control. Values and error bars indicate means ± s.e.m. of three independent experiments.