This protocol explains how to design and clone the guide RNA target sequence into a L1 plasmid ready to accept the gRNA by cloning with BbsI. L1 plasmids are L1_lacZgRNA-Ck2 and L1_lacZgRNA-Ck3. If one gRNA target sequence is cloned into the Ck2 plasmid and another one into Ck3 one, the two L1_gRNA transcription units can be combined with an antibiotic resistance transcription unit and a MpEF1α:Cas9 transcription unit via L2 SapI Loop assembly. This allows for dual gRNA editing.

This protocol describes a Marchantia polymorpha specific modification of the Xie et al 2015 protocol(https://doi.org/10.1073/pnas.1420294112) for the synthesis of multicomplex tRNA-gRNA modules by Golden Gate Assembly/Loop Assembly. It has 3 main steps: A. Primer design and PCR amplification of tRNA-gRNA fragments using the pGTR plasmid as the template. B. Loop assembly cloning of the amplified tRNA-gRNA fragments into an L1 vector,to combine them with the MpU6 promoter C. Loop assembly cloning into the L2 pCsA acceptor vector to combine the MpU6::tRNA-gRNA unit with the transcription unit for Cas9 expression

Protocol for cloning of DNA L0 parts into pUAP4 using SapI type IIS assembly. A standarised L0 part has at its end 5' and 3' fusion sites that follow the common syntax. For Marchantia L0 parts, we commonly use eight positions and nine fusion sites, by combining positions A2-A3 for proximal promoter (PROMP), and B1-B2 for 5’ untranslated region (5UTR). The other types of parts are: A1 for distal promoter (PROMD), B3 for coding sequence with start codon and no stop codon (CDS1), B4 for coding sequence without start or stop codon (CDS2), B5 coding sequence without start codon and with stop codon (CTAG), B6 for 3’ untranslated region (3UTR) and C1 for transcription terminator (TERM). Parts can span multiple fusion sites, like A1-A3 for promoter (PROM), A1-B2 for promoter with 5’ UTR (PROM5), B3-B4 for coding sequence with start codon and no stop codon for N-terminal fusion with CTAG (CDS12), B3-B6 for coding sequence with start and stop codons (CDS), or B6-C1 for 3’ UTR with terminator (3TERM).

A modification of the Ishizaki et al 2008 Agrobacterium mediated Marchantia sporeling transformation protocol is used. Sterilised spores are grown for 5-7 days in ½ strength Gamborg plates prior to co-cultivation for 2 days with agrobacterium in liquid media in multiwell plates. Sporelings are then spread on media with the appropriate selective antibiotic. In about 5 days, positive transformants start to emerge.

Protocol based on: Pollak B, Cerda A, Delmans M, et al (2019) Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytol 222:628–640 https://doi.org/10.1111/nph.15625

This protocol is a modifiction of the ISOLATION OF MAIZE CHLOROPLASTS FOR PROTEIN IMPORT STUDIES protocl from Mark Settles (modified from Ken Cline).

Domestication and cloning of L0 parts into pUAP4 for Loop type IIS assembly

This protocol allows for quick and dirty genomic DNA extraction. It can easily be used for genotyping with PCR. The quality of the genomic DNA extracted is not suitable for any other application.

sites is necessary when a DNA sequence of interest contains an internal BsaI or SapI recognition site.For domestication, overlapping PCR can be protocols.

Design of primers for cloning of L0 parts into pUAP4 vectors.(A) To clone a standardizsed L0 part flanked by the 5' and 3' common syntax fusion sites specific to a type of part, the fusion sites are added to the DNA part by PCR, using specially designed primers.Primers include, in Summary of primers and cloning into pUAP4 protocols.

Protocol based on: Pollak B, Cerda A, Delmans M, et al (2019) Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytol 222:628–640 https://doi.org/10.1111/nph.15625

Protocol based on Pollak B, Cerda A, Delmans M, et al (2019) Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytol 222:628–640 https://doi.org/10.1111/nph.15625

sporangia per transformation planned protocols.

Protocol based on Pollak B, Cerda A, Delmans M, et al (2019) Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytol 222:628–640 https://doi.org/10.1111/nph.15625

gRNA design and cloning with SapI into Loop plasmid L2_lacZgRNA-Cas9-CsA Forked from gRNA design and cloning into Loop L2 plasmids (L2_gRNA-Cas9-CsA and L2_gRNA-CsA plasmids) Eftychis Frangedakis , marta tomaselli , Susana Sauret-Gueto Plant Sciences, University of Cambridge, OpenPlant Works for me dx.doi.org/10.17504/protocols.io.93wh8pe Susana Sauret-Gueto Plant Sciences, University of Cambridge, OpenPlant Dec 04, 2019 1 1 1 1

A modification of the Ishizaki et al 2008 Agrobacterium mediated Marchantia sporeling transformation protocol is used. Sterilised spores are grown for 5 days in 0.5x strength Gamborg plates prior to co-cultivation for 2 days with agrobacterium in liquid media in multiwell plates. Sporelings are then spread on media with the appropriate selective antibiotic. In about 7 days, positive transformants start to emerge.

This protocol explains how to design clone the guide RNA (gRNA) sequence into the L2 plasmids ready to accept the gRNA sequence by cloning with SapI. We have two versions of the plasmid, with or without Cas9: the L2_gRNA-Cas9-CsA and L2_gRNA-CsA plasmids. With L2_gRNA-Cas9-CsA you transform wild-type sporelings. With L2_gRNA-CsA you transform sporelings of a line expressing Cas9.