Primer Sequences.

<p>Primer sequences for sgRNA synthesis, sequencing, and mutagenesis are shown.</p><p>Primer Sequences.</p

Generation of indels and donor DNA insertion following sgRNA guide D cleavage.

<p>The results of single guide D editing in AB6 mice (Experiment 5) with a donor template are depicted. Mice are numbered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g002" target="_blank">Fig. 2b</a>. The B6 allele is indicated with the positions of guides B and D as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g001" target="_blank">Fig. 1</a>. Sequencing results from unaltered <i>Tyr</i> alleles are not shown (5.4.2, 5.5.2, 5.7.2). Deleted nt (-). Inserted nt in brackets.</p

Clustered distribution of deleted nucleotides by CRISPR-Cas9-mediated genome editing.

<p>The number of times a particular nt was found to be deleted upon sequence analysis is represented by the bar above the specific nt. The DNA sequence indicates the PAM sites in red with the sgRNA recognition site for guides B and D in a box, with the guide identity indicated by the arrow below it. For each guide, the predicted cleavage site of Cas9 is indicated as the site between the two red bars. <b>(a)</b> An analysis of all deleted regions from sequence alleles containing one or two disrupted sites without the loss of the intervening residues. <b>(b)</b> As in <b>(a)</b>, but with all alleles (including intervening deletions) plotted. The data represent sequence results of 47 alleles from the 29 genome edited mice with indels.</p

Successful genome editing of <i>Tyr</i> is evident on the day of birth and confirmed by DNA sequencing.

<p><b>(a)</b> Phenotypic results of guide B editing of B6xAB6 fertilized eggs (Experiment 1) resulted in loss of eye pigmentation in two of three pups photographed on day 1 after birth, whereas one pups appeared unaltered (middle pup). Since sequencing analysis was not pursued in these pups, a disruptive indel on the AB6 allele and/or an in-frame indel on the B6 allele could also result in the same phenotypic finding. The mice in Experiment 1 did not survive because the cage housing the pups from guide B was flooded while pups from guide D were cannibalized by the mother. <b>(b)</b> Sequencing results of guide D editing from Experiment 1 are shown here. The <i>Tyr</i> alleles from guide B targeting were not analyzed. The B6 allele is indicated with the position of guide D as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g001" target="_blank">Fig. 1</a>. Mice are numbered with the following convention: (experiment#.mouse#.allele#). Mouse 1.1 (in-frame deletion on the B6 allele) and mouse 1.4 (no indels observed) had black eyes while the remaining mice had pink eyes at birth. Sequencing results from unaltered <i>Tyr</i> alleles are not shown. Deleted nt (-). Inserted nt in brackets.</p

Successful gene repair of point mutation by phenotype and DNA sequencing.

<p><b>(a)</b> Phenotypic results of guide B and D editing of AB6xAB6 fertilized eggs (Experiment 3) resulted in a gain of black coat color in one of three pups photographed on day 5 after birth. Mice 3.1 (black pup), 3.2, and 3.3 are shown. <b>(b)</b> The results of combined guide B and D editing in AB6 mice with a donor template are depicted. When a modified donor was used, the mutations are indicated by (*) above the lowercase base substitutions. Mice are numbered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g002" target="_blank">Fig. 2b</a>. The AB6 allele is indicated with the positions of guides B and D as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g001" target="_blank">Fig. 1</a>. Sequencing results from the unaltered <i>Tyr</i> allele 3.19.2 is not shown. Mouse 3.3 is a genetic but not phenotypic mosaic. Underlined alleles were from a D10A Cas9 injection. Deleted nt (-). Inserted nt in brackets. The introduction of a novel <i>Sal</i>I restriction enzyme site is identified by a box for allele 3.1.1.</p

Paired sgRNAs result in gene disruption by phenotype and DNA sequencing.

<p><b>(a)</b> Phenotypic results of guide B and D editing of B6xB6 fertilized eggs (Experiment 2) resulted in loss of black coat color in two of four pups photographed on day 14 after birth. The following mouse numbers and their phenotypes are shown: 2.3 (black), 2.4 (black), 2.5 (white), 2.6 (white). <b>(b)</b> Sequencing results of combined guide B and D editing are depicted. The B6 allele is indicated with the positions of guides B and D as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g001" target="_blank">Fig. 1</a>. Mice are numbered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g002" target="_blank">Fig. 2b</a>. Sequencing results from unaltered <i>Tyr</i> alleles are not shown (2.4.2, 2.10.2). No PCR product was detected for allele 2.6.2; later it was confirmed to be a large 42 kb deletion (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g008" target="_blank">Fig. 8</a>). Underlined alleles were from a D10A Cas9 injection. Deleted nt (-). Inserted nt in brackets.</p

Indel frequency in Cas9-modified mice does not correlate with survival.

<p>The number of pups with indels, regardless of survival, is plotted as a frequency per injection. All mice that did not survive to at least 3 weeks were categorized as “dead”. A total of 16 independent injections (dead = 10, alive = 6) with range, mean, and SEM are plotted. <i>p</i> = 0.28, Two-tailed Student’s <i>t</i>-test. ns: non-significant.</p

Homologous recombination (HR) in founder mice.

<p><b>(a)</b> Genomic DNA was amplified with primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.t004" target="_blank">Table 4</a>) designed to flank the <i>Tyr</i> mutation site resulting in a PCR product of 1555bp (solid arrow). Amplification of mice 3.1 and 4.9 yielded multiple smaller molecular weight bands. The bands represented by asterisks have been confirmed by sequence analysis and represent an alternate truncated allele. Interestingly, the phenomenon of heteroduplex formation (arrowheads) is also evident in mice with more than one allele size. These artifacts are thought to occur upon heterologous binding of DNA strand from each allele (Thompson et al. 2002). Sanger sequencing of predicted heteroduplex bands confirmed the mixture of both higher and lower molecular weight bands in the product. To eliminate interference with the restriction analysis, the 1555bp band was excised from the gel for all of these mice and subjected to <i>Sal</i>I digest <b>(b)</b>. <b>(c)</b> Black-eyed progeny from mouse 4.9 all appeared to be positive for donor insertion. The predicted digestion products of 835bp and 720bp are indicated by the dashed arrow. The 100bp molecular weight marker was loaded in the left-most lane of each agarose gel with sizes as indicated (in Kb). B6: C57BL/6, AB6: C57BL/6 Albino. Mouse numbers are consistent with the text.</p

Mosaicism following CRISPR-Cas9-mediated genome-editing.

<p><b>(a)</b> Mouse 4.9 is shown in three photographs (top) along with a WT B6 littermate (bottom) at four weeks of age. The patchy coat color distribution was evident several days after birth and has remained consistent throughout development. <b>(b)</b> The results of combined guide B and D editing in B6 mice with a donor template are depicted. The mutations present in the modified donor are indicated by (*) above the lowercase base substitutions. Mice are numbered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g002" target="_blank">Fig. 2b</a>. The B6 allele is indicated with the positions of guides B and D as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g001" target="_blank">Fig. 1</a>. Sequencing results from unaltered <i>Tyr</i> alleles are not shown (4.1.2, 4.4.2, 4.8.3). No PCR product was detected for allele 4.7.2 suggesting a large indel. Deleted nt (-). Inserted nt in brackets.</p

Tyrosinase gene and sgRNA placement.

<p>A representation of the WT B6 <i>Tyr</i> (NM_011661.4) coding sequence with the five exons (numbered) and flanking untranslated regions in gray. The region of interest for genome editing is enlarged. The SNM resulting in an eye and coat color change is indicated (*) and corresponds to nt 230 with reference to the translation start site. The amino acids encoded are shown below the DNA sequence and the critical “DDRE” motif for <i>Tyr</i> function is boxed. Four sgRNAs were designed flanking or including this site with indicated orientations (guides A, B, C, and D). The sgRNA binding sites on the homologous DNA are indicated in red, with the PAM sites underlined.</p