Vyas H et al. (2022), Generation of a homozygous mutant drug transpor...

ECB-ART-50292

Development 2022 Jun 01;14911:. doi: 10.1242/dev.200644.

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Generation of a homozygous mutant drug transporter (ABCB1) knockout line in the sea urchin Lytechinus pictus.

Vyas H , Schrankel CS , Espinoza JA , Mitchell KL , Nesbit KT , Jackson E , Chang N , Lee Y , Warner J , Reitzel A , Lyons DC , Hamdoun A .

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Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources.

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	Fig. 1. Generation of a homozygous mutant drug transporter ABCB1â/â line in the sea urchin Lytechinus pictus. (A) The gene locus of Lp-ABCB1. The sgRNA target sites (maroon triangles) for generating large deletions between exons 10 and 11 are indicated. (B) Predicted Lp-ABCB1 protein topology. Target site regions in the nucleotide binding domains (NBDs, blue ovals) are shown. NBDs are necessary for transport. (C) Schematic of CRISPR/Cas9 gene editing and propagation of Lp-ABCB1 mutant generations. Large deletions are generated in the F0 generation. An F0Ãwild-type outcross creates a heterozygous ABCB1 F1 generation, and the F1 in-cross generates homozygous mutants in the F2 generation. Phenotype analysis uses a calcein-AM (CAM) substrate accumulation assay to quantify the level of ABCB1 transporter activity in each generation.
	Fig. 2. Identification and propagation of an ABCB1Î800âbp deletion germline mutant. (A) Crispant sequence analysis. Genomic DNA was extracted, amplified and cloned from somatic tissue (tube feet) and gametes from metamorphosed juveniles of the F0 (i) generation, and from individual F2 larvae (ii). Blue text, sgRNA target sites; gray boxes, PAM site; magenta dashes, indels. Total sizes of indels are indicated for each exon. (B,C) PCR screening of individual F2 ABCB1Î800 larvae. Wild type (+/+), and heterozygous (+/â) and homozygous (â/â) mutants were identified by gel band pattern (B). Genotypes from individual F2 ABCB1Î800 larvae (n=119) demonstrate near Mendelian inheritance of the mutant alleles (C).
	Fig. 3. A significant loss of transporter efflux activity is present within ABCB1Î800 F2 line larvae. (A,B) CAM substrate accumulation assay. ABCB1Î800 F2 line larvae exhibit higher intracellular levels of the ABCB1 substrate CAM (magenta; A) when compared with outbred wild-type larvae (B; ****P<0.0001, unpaired two-tailed MannâWhitney t-test). Micrograph examples of low, medium and maximum intracellular CAM accumulation in ABCB1Î800 line F2 larvae are shown (A). DIC, differential interference contrast. Scale bars: 50âÂµm. Data are pooled from three independent mate pairs and all images were acquired and processed using the same settings. Representative wild-type images are shown in Fig.Â S7. (C) The distribution of CAM accumulation is broader within ABCB1Î800 line F2 larvae. Raw fluorescence values were normalized to the maximum accumulation values. Frequency distribution of normalized values shows a shift in peak fluorescence evident in ABCB1Î800 F2 line larvae when compared with outbred wild-type larvae. (D) Homozygous F2 ABCB1Î800â/â mutants survive metamorphosis. A representative 1-month-old juvenile is shown.

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