Protocol for Transfection of Bodo saltans with SaCas9 RNP complex in conjunction with eGFP-NEO plasmid by electroporation

Fatma Gomaa, Zhu-Hong Li, Roberto Docampo, Virginia Edgcomb

Published: 2021-08-13 DOI: 10.17504/protocols.io.bxbwpipe
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Abstract

Developing transfection protocol for Bodo saltans , using SaCas9/sgRNA ribonucleoprotein (RNP) complex in conjunction with DNA repair template to disrupt the Paraflagellar rod 2 gene ( BsPFR2 ) 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 eGFP fused with the drug selection gene nptII/neo and flanked by 500 bp of the untranslated regions (UTRs) upstream and downstream of the targeted BsPFR2 as homologous repair arms.

Steps

1.

Step 1: Plasmid construction to target the PFR-2 gene

-A 2512 bp promoter-less cassette is designed to target and knock out the B. 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).

2.

Step 2: Co-delivery of SaCas9 RNP complex and the DNA repair template for BsPFR2 disruption

-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

2.1.

-Table 1: NEPA 21 electroporation parameters used in our study

ABCDEFGHIJKLM
Poring pulse********Transfer pulse********
****VPD (ms)PI (ms)Ndecay ratepolarityVPD (ms)PI (ms)Ndecay ratepolarity
1200250110%+6099999540%+/-
299550710%+995050540%+/-
3150550310%+5010999540%+/-

-V: voltage strength; PD: pulse duration; PI: pulse interval; N: number of pulses

2.2.
Figure 1: Illustration of results obtained from transfection of B. saltans with Cas9RNP complex along with DNA repair cassette. A: Schematic representation of the PFR-2 locus into the genome of B. saltans. the targeted locus of the Cas9RNP complex and the PCR primers annealing sites; B:Schematic representation of the double stranded DNA repair template and the PCR primers annealing sites; C: Schematic representation showing that the construct replace the endogenous BsPFR2 with eGFP-NEO cassette by double cross-over homologous recombination, PCR#1 gel image and diagram showing the on-target integration of the plasmid in transfected cell at the 3’ flanking region PCR with a PCR product at 0.8 kb; PCR#3 image and diagram showing the integration of the plasmid at the 5’ flanking region with PCR product at 2.6 kb; D: Schematic representation showing that the construct replace the endogenous BsPFR2 with eGFP-NEO cassette by double cross-over homologous recombination, with an insertion of additional 600 bp of the PFR-2 resulted from cross-over event, with PCR# 2 gel image showing a PCR product at 1.4 kb.
Figure 1: Illustration of results obtained from transfection of B. saltans with Cas9RNP complex along with DNA repair cassette. A: Schematic representation of the PFR-2 locus into the genome of B. saltans. the targeted locus of the Cas9RNP complex and the PCR primers annealing sites; B:Schematic representation of the double stranded DNA repair template and the PCR primers annealing sites; C: Schematic representation showing that the construct replace the endogenous BsPFR2 with eGFP-NEO cassette by double cross-over homologous recombination, PCR#1 gel image and diagram showing the on-target integration of the plasmid in transfected cell at the 3’ flanking region PCR with a PCR product at 0.8 kb; PCR#3 image and diagram showing the integration of the plasmid at the 5’ flanking region with PCR product at 2.6 kb; D: Schematic representation showing that the construct replace the endogenous BsPFR2 with eGFP-NEO cassette by double cross-over homologous recombination, with an insertion of additional 600 bp of the PFR-2 resulted from cross-over event, with PCR# 2 gel image showing a PCR product at 1.4 kb.

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