Objective: Convert a hulled (covered) barley into a hull-less (Naked Objective: Convert a hulled (covered) barley into a hull-less (Naked!) barley Taketa PNAS 2008

Hulled phenotype controlled by one gene: NUD >NUD_CDS ATGGTACAGTCCAAGAAGAAGTTTCGCGGCGTCAGGCAGCGCCACTGGGGCTCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGCGGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCAACGGGGAGATCATCGTCGCCCCAGCAGCAGCAGCACGGGACATTCGCGGTGGCGTTGGCTCGTCGTCCTCCGGGGCCGCCGGCGCCAGCAGCCTGTCACAGATCCTCAGCGCCAAGCTCCGCAAGTGCTGCAAGACACCGTCCCCGTCCCTCACCTGCCTCCGCCTCGACACCGAGAAGTCCCACATTGGCGTCTGGCAGAAGCGCGCGGGTGCCCGTGCCGACTCCAGCTGGGTCATGACCGTCGAGCTCAACAAGGAGCCGGCCGCAGCGGCACCACCAACGCCCAGCGACAGCACGGTGTCGGCGACTCCTTCCTCGTCCACGTCCACGTCCACAACGGGCTCCCCACCGGAGGCAATGGAGGACGAAGAGAGGATCGCGCTGCAGATGATAGAGGAGCTGCTGAGCAGGAGCAGCCCGGCTTCGCCGTCACATGGGCTGCTGCACGGTGAAGAAGGCAGCCTCCTCATCTGA Disrupting this gene, through inducing deletions or mutations, should cause loss of the hulled phenotype

RNA-guided Cas9-mediated genome engineering is used to create double stranded breaks Cas9 is guided to specific DNA sequences by a so-called “single guide RNA” (sgRNA) sgRNA guide target sequence must be followed by a “Protospacer Adjacent Motif” (PAM), consisting of NGG Any 23bp sequence ending ‘GG’ can be targeted by an RNA-guided Cas9

5’ GNNNN NNNNN NNNNN NNNNN NGG 3’ Designing the sgRNA Target needs to start with G as the chosen promoter start of transcription is G PAM sequence (NGG) needs to follow target 5’ GNNNN NNNNN NNNNN NNNNN NGG 3’ Target sequence 20bp

5’ GNNNN NNNNN NNNNN NNNNN NGG 3’ Designing the sgRNA For testing transgenics, it is useful to have a restriction site spanning the site of the double stranded break 5’ GNNNN NNNNN NNNNN NNNNN NGG 3’ Mutations or deletions following the double stranded break induced by Cas9 will disrupt the restriction site

Step 1: Select targets within NUD NGG Target 1 Target 2 Target 3 Target 4 Target 5 Target 6 … GACGCCGCGAAACTTCTTCTTGG GCGGCGTCAGGCAGCGCCACTGG GGCGTCAGGCAGCGCCACTGGGG GGCAGCGCCACTGGGGCTCCTGG GCAGCGCCACTGGGGCTCCTGGG GCTCCTGGGTCTCCGAGATCAGG … Python script: identify PAM sequences take 20bp before check begins with G Manual curation: check targets for restriction sites 20bp Multiple targets can be chosen to create multiple sgRNAs

Expected deletion in bold Step 1: Chosen targets Restriction sites Target 1 PAM target Target 2 PAM target >NUD_CDS ATGGTACAGTCCAAGAAGAAGTTTCGCGGCGTCAGGCAGCGCCACTGGGGCTCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGCGGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCAACGGGGAGATCATCGTCGCCCCAGCAGCAGCAGCACGGGACATTCGCGGTGGCGTTGGCTCGTCGTCCTCCGGGGCCGCCGGCGCCAGCAGCCTGTCACAGATCCTCAGCGCCAAGCTCCGCAAGTGCTGCAAGACACCGTCCCCGTCCCTCACCTGCCTCCGCCTCGACACCGAGAAGTCCCACATTGGCGTCTGGCAGAAGCGCGCGGGTGCCCGTGCCGACTCCAGCTGGGTCATGACCGTCGAGCTCAACAAGGAGCCGGCCGCAGCGGCACCACCAACGCCCAGCGACAGCACGGTGTCGGCGACTCCTTCCTCGTCCACGTCCACGTCCACAACGGGCTCCCCACCGGAGGCAATGGAGGACGAAGAGAGGATCGCGCTGCAGATGATAGAGGAGCTGCTGAGCAGGAGCAGCCCGGCTTCGCCGTCACATGGGCTGCTGCACGGTGAAGAAGGCAGCCTCCTCATCTGA Expected deletion in bold

Overview of Golden Gate assembly Golden Gate Assembly allows for the efficient assembly of DNA fragments using sequential or simultaneous digestion and ligation reactions. A Type IIS restriction enzyme, such as BsaI, creates DNA fragments with unique overhangs, and a T4 DNA ligase is used to assemble the fragments together. New England BioLabs https://www.neb.com/products/e1600-neb-golden-gate-assembly-mix#Product%20Information

Complementary sequence to anneal to sgRNA plasmid Step 2: Synthesize chosen primers for Golden Gate assembly Target sequence 20bp Forward tgtggtctcaATTGNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaag Reverse tgtggtctcaAGCGTAATGCCAACTTTGTAC Digestion site (BsaI) Complementary sequence to anneal to sgRNA plasmid Additional flanking sequence to target is for use as primer for amplification from plasmid note: PAM site is not part of the target sequence

Step 3: Amplify a unique sgRNA using primer pair Amplify from plasmid containing the sgRNA scaffold PCR product: BsaI site Target sequence 20bp tgtggtctcaATTGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTACGCTtgagaccaca BsaI site (reverse complement) Overhang sequences used for Golden Gate Assembly

ALTERNATIVELY (combine step 2 and 3) Synthesize the whole sgRNA including desired target sequence PCR product: BsaI site Target sequence 20bp tgtggtctcaATTGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTACGCTtgagaccaca BsaI site (reverse complement) Overhang sequences used for Golden Gate Assembly

Set up digestion-ligation reaction with: Step 4: Assemble level 1 sgRNA expression cassette using Golden Gate Assembly Set up digestion-ligation reaction with: Plasmid containing Level 0 wheat U6 promoter PCR amplicon Ta Level 1 acceptor plasmid NB. This step can be repeated to create multiple sgRNAs

Initial plasmid containing U6 promoter from wheat Step 4: Assemble level 1 sgRNA expression cassette using Golden Gate Assembly Level 0 Initial plasmid containing U6 promoter from wheat Ta

LacZ can be used as selectable marker as should be swapped for insert Step 4: Assemble level 1 sgRNA expression cassette using Golden Gate Assembly Level 1 Acceptor plasmid pICH47732 Description: Position 1 Comments: AddGene #48000 Inside overhang L: GGAG Inside overhang R: CGCT Resistance: Amp LacZ can be used as selectable marker as should be swapped for insert

plasmid containing the sgRNA scaffold Recap Forward primer plasmid containing the sgRNA scaffold Plasmid containing Level 0 wheat U6 promoter Reverse primer Ta PCR amplicon Level 1 acceptor plasmid

Multiple sgRNAs can be added Step 5: Assemble Level 2 Assembled sgRNAs are combined with a selectable marker and a Cas9 cassette into a Level 2 Acceptor. This creates an expression cassette ready for transformation into Agrobacterium, and subsequent barley transformation. Selectable marker Cas9 cassette Multiple sgRNAs can be added Step 6: Transform Agrobacterium with final level 2 construct

The transformed Agrobacterium is dropped on to the embryo Step 7: Transformation of barley Immature embryos are harvested from immature seed and their embryonic axis is removed The transformed Agrobacterium is dropped on to the embryo Harwood et al. (2009) Barley Transformation Using Agrobacterium-Mediated Techniques

Transformation of barley Embryos transferred to callus induction plates Plated on regeneration media containing selection Transgenic barley transferred to culture tube Plants left to develop before potting into soil Harwood et al. (2009) Barley Transformation Using Agrobacterium-Mediated Techniques

Step 8: Confirmation of transgenic barley Carry out DNA extraction of transgenics PCR amplify the site of sgRNA target using flanking markers sgRNA 1 sgRNA 2 Forward primer Reverse primer >NUD_CDS ATGGTACAGTCCAAGAAGAAGTTTCGCGGCGTCAGGCAGCGCCACTGGGGCTCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGCGGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCAACGGGGAGATCATCGTCGCCCCAGCAGCAGCAGCACGGGACATTCGCGGTGGCGTTGGCTCGTCGTCCTCCGGGGCCGCCGGCGCCAGCAGCCTGTCACAGATCCTCAGCGCCAAGCTCCGCAAGTGCTGCAAGACACCGTCCCCGTCCCTCACCTGCCTCCGCCTCGACACCGAGAAGTCCCACATTGGCGTCTGGCAGAAGCGCGCGGGTGCCCGTGCCGACTCCAGCTGGGTCATGACCGTCGAGCTCAACAAGGAGCCGGCCGCAGCGGCACCACCAACGCCCAGCGACAGCACGGTGTCGGCGACTCCTTCCTCGTCCACGTCCACGTCCACAACGGGCTCCCCACCGGAGGCAATGGAGGACGAAGAGAGGATCGCGCTGCAGATGATAGAGGAGCTGCTGAGCAGGAGCAGCCCGGCTTCGCCGTCACATGGGCTGCTGCACGGTGAAGAAGGCAGCCTCCTCATCTGA

Run PCR products on gel against wild type control WT 1 2 If see PCR product from transgenic samples is smaller than wild type, then deletion of sequence has occurred. WT 1 2 If bands similar size, deletion or mutation is not easy to detect. Then need to digest the PCR product.

Digest PCR sample using restriction enzymes Restriction sites PAM target PAM target BgII >NUD_target_region ATGGTACAGTCCAAGAAGAAGTTTCGCGGCGTCAGGCAGCGCCACTGGGGCTCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGCGGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCA

Digest PCR sample using restriction enzymes WT 1 2 Digestion with BgII PCR product will not be digested if there is disruption of the restriction site. Therefore, the gene has been disrupted. BgII >NUD_target_region ATGGTACAGTCCAAGAAGAAGTTTCGCGGCGTCAGGCAGCGCCACTG GGGCTCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGCGGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCA Confirmed transgenics can be used for experimentation