Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest

Deep sequencing reveals high mutagenesis rates for acute multi-sgRNA CRISPR/Cas9 KO of <i>osgep</i> and <i>tprkb</i>.

<p>(<b>a</b>) Mutation rates per sgRNA (i.e. likelihood of generating at least one mutation on each allele), given hypothetical mutation rates for 3 different sgRNAs. Note that the achieved likelihood for occurrence of at least one mutation per allele is high (92.16%), even though mutation rates for each sgRNA are moderate (50–80%). (<b>b-e</b>) Deep sequencing reveals high mutagenesis rates for most sgRNAs and sgRNA dependent frameshift rates. For each gene, deep sequencing data of 96 larvae at 48 hpf were analyzed individually using the tool CRISPResso [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191503#pone.0191503.ref024" target="_blank">24</a>]. (<b>b</b>) For <i>osgep</i>, median mutagenesis rate was 69.3% for sgRNA1, 93.0% for sgRNA2, and 79.6% for sgRNA3. (<b>c</b>) For all mutated <i>osgep</i> alleles, the median fractions of frame shifts were 56.7% for sgRNA1, 44.7% for sgRNA2, and 61.0% for sgRNA3. (<b>d</b>) For <i>tprkb</i>, median mutagenesis rate was 100% for sgRNA1, 92.3% for sgRNA2, and 41.0% for sgRNA5. (<b>e</b>) For all mutated <i>tprkb</i> alleles, the median fractions of frame shifts were 44.3% for sgRNA1, 81% for sgRNA2, and 52.8% for sgRNA5.</p

Likelihoods for at least one mutation on each allele and at least one frameshift mutation on each allele are clearly improved in acute multi-sgRNA CRISPR/Cas9 KO of <i>osgep</i> and <i>tprkb</i>.

<p><b>(a-b)</b> The likelihood is shown of generating at least one mutation on each allele <i>P(M)</i> (<b>a</b>) and of generating at least one frameshift mutation on each allele <i>P(F)</i> (<b>b</b>), where <i>M</i> = at least one mutation on each allele, <i>F</i> = at least one frameshift mutation on each allele, <i>q</i> = probability of no mutation, <i>r</i> = specific sgRNA, <i>im</i> = fraction of in-frame mutations of all mutations, <i>nm</i> = fraction of non-coding mutations of all mutations. (<b>c-h</b>) The likelihoods of at least 1 mutation on each allele and at least 1 frameshift mutation on each allele were calculated based on observed mutagenesis and frameshift rates for 96 individual fish per gene according to the equation in (<b>a</b>) and (<b>b</b>). (<b>c</b>) The individual analysis for <i>osgep</i> revealed a median <i>P(M)</i> of 48.1% for sgRNA1, 86.6% for sgRNA2, and 63.4% for sgRNA3. (<b>d</b>) The median <i>P(F)</i> was 15.1% for sgRNA1, 14.6% for sgRNA2, and 18.5% for sgRNA3. (<b>e</b>) For the pooled <i>osgep</i> sgRNAs, the median <i>P(M)</i> was 99.3%, and 64.2% for <i>P(F)</i>. (<b>f</b>) The individual analysis for <i>tprkb</i> revealed a median <i>P(M)</i> of 100% for sgRNA1, 84.7% for sgRNA2, and 16.9% for sgRNA5. (<b>g</b>) The median <i>P(F)</i> was 15.5% for sgRNA1, 52.2% for sgRNA2, and 4.1% for sgRNA5. (<b>h</b>) For the pooled <i>tprkb</i> sgRNAs, the median <i>P(M)</i> was 100%, and 78.8% for <i>P(F)</i>.</p

Acute multi-sgRNA CRISPR/Cas9 knockout reproduces the microcephaly phenotype of stable <i>osgep</i> and <i>tprkb</i> KO lines.

<p>Larvae were imaged from a dorsal view. Head diameter to total body length was calculated. This ratio was normalized by the mean of the uninjected/wildtype control and defined as “microcephaly index” (MI). MI was determined at 6 dpf except for the stable KO line osgep c.102_105del at 4 dpf due to early lethality (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191503#pone.0191503.g004" target="_blank">Fig 4a</a>). (<b>a</b>) Representative phenotypes of a larva that was injected with the <i>osgep</i> targeting multi-sgRNA pool compared to a scrambled control larva. The red lines display the typical axes used for the measurements. (<b>b</b>) For <i>osgep</i>, the acute multi-sgRNA KO showed a significant microcephaly compared to uninjected and scrambled control. (<b>c</b>) The microcephaly phenotype is recapitulated in the homozygous larvae of the stable KO line <i>osgep c</i>.<i>102_105del</i> compared to wildtype and heterozygous clutch mates. (<b>d</b>) For <i>osgep</i>, MI is significantly higher in acute KO larvae compared to homozygous stable KO larvae. (<b>e</b>) For <i>tprkb</i>, the acute multi-sgRNA KO showed a significant microcephaly compared to uninjected and scrambled control. (<b>f</b>) The microcephaly is recapitulated in the homozygous larvae of the stable KO line <i>tprkb</i> c.370_376delinsAA compared to wildtype and heterozygous clutch mates. (<b>g</b>) For <i>tprkb</i>, no significant difference in MI was found for acute KO larvae compared to homozygous stable KO larvae.</p

Deep sequencing reveals sgRNA dependent indel profiles.

<p>For each gene, deep sequencing data of 96 larvae at 48 hpf were analyzed individually using the tool CRISPResso [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191503#pone.0191503.ref024" target="_blank">24</a>]. (<b>a-f</b>) On the X-axis, the graphs show the numbers of base pairs (bp) deleted (negative numbers) or inserted (positive numbers). The Y-axis displays the fraction of the particular indel of all mutated reads per each one of the 96 individual larvae. As many larvae had more than 1 indel, all fractions give the mean frequency in 96 individual larvae. (<b>a</b>) For <i>osgep</i> sgRNA1, the most frequent deletion was 6 bp in length (23.5%, arrow head), followed by 8 bp (11.1%). The most frequent insertion was 1 bp (6.8%). (<b>b</b>) For <i>osgep</i> sgRNA2, the most frequent deletion was 10 bp (18.2%, arrow head), followed by 2 bp (16.3%). The most frequent insertion was 1 bp (6.3%). (<b>c</b>) For <i>osgep</i> sgRNA3, the most frequent deletion was 10 bp (17.1%, arrow head), followed by 12 bp (10.9%). The most frequent insertion was 1 bp (4.7%). <i>osgep</i> sgRNA2 and sgRNA3 additionally create larger deletions up to 65 bp together (<b>b-c</b>) since they target the same exon. (<b>d</b>) For <i>tprkb</i> sgRNA1, the most frequent deletion was 3 bp in length (22.9%, arrow head), followed by 6 bp (13.5%). The most frequent insertion had a length of 1 bp (3.9%). (<b>e</b>) For <i>tprkb</i> sgRNA2, the most frequent deletion was 2 bp (23.8%, arrow head), followed by 1 bp (15.3%). The most frequent insertion was 1 bp (3.1%). (<b>f</b>) For <i>tprkb</i> sgRNA5, the most frequent deletion was 3 bp (26.9%, arrow head), followed by 2 bp (10.0%). The most frequent insertion was 7 bp (2.6%). (<b>g</b>) For <i>osgep</i>, deletions accounted for 77.9% of all mutations for sgRNA1, 82.8%for sgRNA2 and 83.6%. for sgRNA3, compared to 22.1% insertion for sgRNA1, 17.2% for sgRNA2 and 16.4% for sgRNA3. (<b>h</b>) For <i>tprkb</i>, deletions accounted for 73.8% of all mutations for sgRNA1, 83.4%for sgRNA2 and 82.5%. for sgRNA5, compared to 26.2% insertion for sgRNA1, 16.6% for sgRNA2 and 17.5% for sgRNA5.</p

Acute multi-sgRNA CRISPR/Cas9 KO and stable KO lines for <i>osgep</i> and <i>tprkb</i> demonstrate reduced survival.

<p>In order to generate survival curves, larvae were monitored twice a day for 22 days and transferred to rotifer feeding solution at 8 dpf. Tracing started at 24 hpf. (<b>a</b>) The acute multi-sgRNA KO for <i>osgep</i> shows an increased death rate as of 10 dpf with a survival of 42.9% at 22 dpf compared to scrambled and uninjected control with both survival of 73.4% at 22 dpf (P<0.0001). (<b>b</b>) In the stable <i>osgep</i> KO line with the truncating mutation <i>c</i>.<i>102_105del</i>, homozygous larvae show complete mortality by 5 dpf, whereas 71% of the heterozygous and 80% of the wildtype larvae survive until the end of the observation at 21 dpf. No significant difference in survival was found between heterozygous and wildtype larvae (P = 0.3269). (<b>c</b>) For <i>osgep</i>, homozygous stable KO larvae show a significantly increased and earlier mortality compared to acute KO larvae (P<0.0001). (<b>d</b>) The acute multi-sgRNA KO for <i>tprkb</i> shows an increased death rate as of 12 dpf with a survival of 19.4% at 22 dpf compared to scrambled (58.0%) and uninjected control (63.8%) at 22 dpf (P<0.0001).</p

Mutations in multiple components of the nuclear pore complex cause nephrotic syndrome

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained, Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype