Further analysis of barley MORC1 using a highly efficient RNA‐guided Cas9 gene‐editing system

Summary Microrchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL‐type ATPase and an S5‐fold. In plants, MORC proteins were first discovered in a genetic screen for Arabidopsis thaliana mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and genome stabilization. Little is known about the role of MORC proteins of cereals, especially because knockout (KO) mutants were not available and assessment of loss of function relied only on RNAi strategies, which were arguable, given that MORC proteins in itself are influencing gene silencing. Here, we used a Streptococcus pyogenes Cas9 (SpCas9)‐mediated KO strategy to functionally study HvMORC1, one of the current seven MORC members of barley. Using a novel barley RNA Pol III‐dependent U3 small nuclear RNA (snRNA) promoter to drive expression of the synthetic single guide RNA (sgRNA), we achieved a very high mutation frequency in HvMORC1. High frequencies of mutations were detectable by target sequencing in the callus, the T0 generation (77%) and T1 generation (70%–100%), which constitutes an important improvement of the gene‐editing technology in cereals. Corroborating and extending earlier findings, SpCas9‐edited hvmorc1‐ KO barley, in clear contrast to Arabidopsis atmorc1 mutants, had a distinct phenotype of increased disease resistance to fungal pathogens, while morc1 mutants of either plant showed de‐repressed expression of transposable elements (TEs), substantiating that plant MORC proteins contribute to genome stabilization in monocotyledonous and dicotyledonous plants.

All vectors contain a ccdB cassette and must be propagated in DB3.1 or ccdB survival cells.
The arrow below the ccdB cassette indicates the direction of the sgRNA array after cloning.

Selection and design of sgRNAs
sgRNA coding sequences are introduced to the pMGE system as 23-24 nt long, hybridized oligonucleotides (oligos). Any PAM sequence (NGG) present in a target region can potentially serve as "anchoring point" for design of an sgRNA, although GC content, on target activity and off-targets should be considered. Note that the PAM sequence is not part of the actual sgRNA, but only present in the target sequence. See for example the following review for a list of tools for selecting target sites: Oligo 1 has to start with gttg for pOsU6 vectors Oligo 1 has to start with cttg for pTaU6 vectors The "G" within the "gttG" or "cttG" used as cloning overhang (Oligo 1) is the transcription start site. sgRNA3 and sgRNA4 are examples for target sites G(N) 19 NGG. In this case, 23 nt oligos may be used, and the variable part of the sgRNA will be 20 nt in length with perfect complementarity. sgRNA1 and sgRNA2 are examples for target sites (N) 20 NGG. In this case, 24 nt oligos should be used. This will result in a sgRNA variable part of 21 nt in length, starting with a non-complementary "G" followed by 20 nt complementary to the target site. To our experience, the "dangling G" when addressing (N) 20 NGG target sites does not have negative effects on Cas9 efficiency, but this was not evaluated in detail.
sgRNA and oligonucleotide design for U3 promoter shuttle vectors: Oligo 1 has to start with agca for pHvU3 vectors Oligo 1 has to start with ggca for pOsU3 vectors The "A" within the "agcA" or "ggcA" used as cloning overhang (Oligo 1) is the transcription start site. sgRNA7 and sgRNA8 are examples for target sites A(N) 19 NGG. In this case, 23 nt oligos may be used, and the variable part of the sgRNA will be 20 nt in length with perfect complementarity. sgRNA5 and sgRNA6 are examples for target sites (N) 20 NGG. In this case, 24 nt oligos should be used. This will result in a sgRNA variable part of 21 nt in length, starting with a non-complementary "A" followed by 20 nt complementary to the target site. To our experience, the "dangling A" when addressing (N) 20 NGG target sites does not have negative effects on Cas9 efficiency, but this was not evaluated in detail.

Not allowed within sgRNA sequences: BsaI sites [GGTCTC], BpiI sites [GAAGAC], polyT stretches [≥ 5 Ts; transcriptional termination]
Note for sgRNA cloning procedures: The overhangs used for cloning of hybridized oligos in vectors containing the OsU6 promoter are not ideal. The sticky ends from BpiI digestion (GTTG/GTTT) can re-ligate at a certain frequency, as there is only one nt mismatch. Normally, ~ 10 % of clones on a plate will be the empty vector without the ccdB cassette. Increasing amount of hybridized oligo may help. We advise use of polyclonal plasmid preparations (see below) when working with shuttle vectors.

Cloning and utilization of "one step" vectors
If desired, M1E modules (containing any U3/U6) promoters can be transferred into "recipient" vectors by BsaI cut/ligation to create "one step, one nuclease" vectors as previously described (pDGE62-65 in Ordon et al., 2017, Plant Journal: Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit). BsaI cut/ligation reations are essentially prepared as described (page 8), but should be terminated by a final cycle of BsaI digestion (after denaturation) to ligate any remaining "recipient" vector. The following steps should be subsequently conducted to load one step vectors with oligonucleotides to generate functional nucleases: 1. Hybridization of oligos