TITLE: Protocol for Transfection of Bodo saltans with SaCas9 RNP complex in conjunction with eGFP-NEO plasmid by electroporation
AUTHORS: Fatma Gomaa, Zhu-Hong Li, Roberto Docampo, Virginia Edgcomb

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Developing transfection protocol for </span><span style = "font-style:italic;">Bodo saltans</span><span>, using SaCas9/sgRNA ribonucleoprotein (RNP) complex in conjunction with DNA repair template to disrupt the Paraflagellar rod 2 gene (</span><span style = "font-style:italic;">BsPFR2</span><span>) and increase the efficiency of targeted homologous recombination when a repair template DNA is provided. The exogenous repair template is double stranded DNA and it consists of </span><span style = "font-style:italic;">eGFP</span><span> fused with the drug selection gene </span><span style = "font-style:italic;">nptII/neo </span><span>and flanked by 500 bp of the untranslated regions (UTRs) upstream and downstream of the targeted </span><span style = "font-style:italic;">BsPFR2</span><span> as homologous repair arms.</span></div><style>
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[STEPS]
?. Step 1: Plasmid construction to target the PFR-2 gene -A 2512 bp promoter-less cassette is designed to target and knock out theB. saltans 69 KDa paraflagellar rod protein 2C (PFR-2), (GenBank accession #CYKH01000743: scaffold1667, positions 3455 to 6406). -This cassette is designed to replace the PFR-2 gene with a fusion of the eGFP (enhanced green fluorescent protein) and Neomycin genes. It contains 500 bp homologous arms at the 5' and 3' ends. -The construct was linearized with the restriction enzyme Xbal (New England BioLabs) prior to electroporation. The plasmid sequence was deposited in GenBank under accession number (MZ522125).
?. Step 2: Co-delivery of SaCas9 RNP complex and the DNA repair template for BsPFR2 disruption 
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							-10 ml of B. saltans cells from cultures at log phase (2-3 days old cultures) were filtered using a 7 micron nylon filter and washed 3 times in ddH2O by centrifugation at 950xg for 4 minutes.-Total cell count used for electroporation was between 1x106 to 2x106-2 µg of sgRNA were annealed with 4 µg of SaCas9 for 5 minutes at 37°C, then this mixture was combined with 50 µg of PFR-GFP-Neo plasmid and incubated 2 minutes at room temperature. -B. saltans cells were electroporated using a square wave electroporator (NEPA21, Bulldog Bio, Inc.), with the electroporation parameters presented in Table 1. -The cells were recovered immediately after electroporation and incubated in B. saltans growth media. -G418 was added to transfected cultures 48 hours post-electroporation at concentration 2 µg/ml and increased gradually to 3 µg/ml over few weeks.-Genotyping analysis using PCR primers sets targets different regions on the plasmid as well as on the genome of B. saltans to confirm the on-target plasmid integration are presented in Figure 1
?. -Table 1: NEPA 21 electroporation parameters used in our study       Poring pulse              Transfer pulse                   V    PD (ms)    PI (ms)    N     decay rate    polarity    V    PD (ms)    PI (ms)    N    decay rate    polarity     1    200    25    0    1    10%    &nbsp;+    60    99    999    5    40%    &nbsp;&nbsp;&nbsp; +/-    2    99    5    50    7    10%    &nbsp;+    99    50    50    5    40%    &nbsp;&nbsp;&nbsp; +/-    3    150    5    50    3    10%    &nbsp;+    50    10    999    5    40%    &nbsp;&nbsp;&nbsp; +/-  -V: voltage strength; PD: pulse duration; PI: pulse interval; N: number of pulses
Poring pulse              Transfer pulse                   V    PD (ms)    PI (ms)    N     decay rate    polarity    V    PD (ms)    PI (ms)    N    decay rate    polarity     1    200    25    0    1    10%    &nbsp;+    60    99    999    5    40%    &nbsp;&nbsp;&nbsp; +/-    2    99    5    50    7    10%    &nbsp;+    99    50    50    5    40%    &nbsp;&nbsp;&nbsp; +/-    3    150    5    50    3    10%    &nbsp;+    50    10    999    5    40%    &nbsp;&nbsp;&nbsp; +/-
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