Sgrnas

The single guide RNAs (sgRNAs; Synthego Corporation, Table 1) were designed using an online designer tool by the Genetic Perturbation Platform (Broad Institute) by submitting three sequences of the LRP2 gene: the transmembrane domain (TMD), asparagine-proline-methionine-tyrosine (NPMY) motif, and proline-proline-proline-serine-proline-serine (PPPSPS) motif (Fig. 1). See the Additional file 1 for details.Table 1

Designed sgRNA sequences

Target	Designed sequence
TMD	CTCCA ATTAC GACGA TCAAG
NPMY motif	CTGTC TCTGG CTGAG TACAT
PPPSPS motif	TTAGC AGGGA GCGAA GGTGA

Fig. 1

Visualization of human megalin structure and designed cuts. Selected sgRNA sequences targeting the transmembrane domain (TMD), motifs important in regulation of function and ligands trafficking (NPMY motif), and basal phosphorylation of the receptor (PPPSPS motif)

Perkinsus marinus cells were prepared following the Cell Line Optimization Nucleofector Kit protocol before electroporation. Using the Nucleofector^TM 2b, 10 μg of Streptococcus pyrogenes Cas9 (SpCas9) nuclease TrueCut^TM Cas9 protein v2 (Thermo Fisher Scientific, Vilnius, Lithuania) and 10 μg of sgRNAs (Synthego, Silicon Valley, CA, United States) were mixed in 100 μl of Lonza’s solution V and incubated at room temperature for 15 min for hybridization of sgRNA and Cas9 protein (Beneke et al., 2017 (link)). Twenty micrograms of dried dDNA plasmid were resuspended with a SpCas9–gRNA complex and electroporated into 25.0 × 10⁶P. marinus trophozoites, using pre-set D-023 program (Fernández Robledo et al., 2008 (link)). Immediately after electroporation, the individual electroporation cuvettes’ contents were transferred to a 12-well plate, each well containing 1 ml of DME:Ham’s F12 (1:2) supplemented with 5% FBS, to allow cells to recover (Gauthier and Vasta, 1995 (link)). Upon identifying the fluorescent cells, the cells were spun down at 1,000 × g for 5 min at room temperature and resuspended into fresh media. Cells were screened for green fluorescence at 24, 72, and 120 h, and 2, 4, and 6 weeks post-transfection using confocal microscopy and flow cytometry.

The Cas9 expression plasmid JDS246 was obtained from Addgene. Single guide RNAs (sgRNAs) were designed using the CRISPOR software (Concordet and Haeussler, 2018 (link)) and synthesized as chemically modified sgRNAs by Synthego (Menlo Park, CA, United States).
Cas9 mRNAs were transcribed in vitro, capped and polyadenylated using the T7 mScript^TM Standard mRNA Production System (C-MSC100625, CELLSCRIPT, Madison, WI, United States). Cas9 mRNA and sgRNA were diluted in RNase-free TE buffer (1 mM Tris–Cl pH 8.0, 0.1 mM EDTA), stored in −80°C in 10 μl aliquots, and were thawed and kept on ice before microinjection.

Cas9-mediated knockout of IGHMBP2 was performed using two sgRNAs (Synthego) to target exon 2 of IGHMBP2 in combination: 5′-CAGAGAGAUGUUCUCCUGCC-3′ and 5′-CUGAAAGAGCUCCAGAGCCG-3′. Ribonucleoprotein (RNP) complexes were prepared by combining 50 pmol of recombinant Cas9 to 50 pmol of each sgRNA and incubating the mixture for 20 min at 37°C. K562 CRISPRi cells (Gilbert et al, 2014 (link)) were nucleofected (SF Cell Line 4D kit; Lonza) with the RNP. Nucleofected clones were isolated by limiting dilution and indel-containing clonal populations were screened by extracting gDNA using Quick Extract (Biosearch Technologies) for PCR amplification using primers targeting up and downstream the Cas9 cut-site: 5′-GGTTGTGGCATTAACTGCCC-3′ and 5′-CCCACATCAATTGTTGGAC-3′. PCR products were separated on a 3% agarose gel at 100 V, and IGHMBP2 gDNA disruption in select clones was further validated via Sanger sequencing with primer 5′-CTTTACGAGGGTACAAGTCACGG-3′ and Western blotting.