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Mouse Snx13 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Snx13 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Snx13 gene (NCBI Reference Sequence: NM_001014973 ; Ensembl: ENSMUSG00000020590 ) is located on Mouse chromosome 12. 26 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 26 (Transcript: ENSMUST00000048519). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Snx13 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-209G1 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Homozygous null mice are growth retarded and die at midgestation with defects in neural tube closure, vasculogenesis and placental development. Mutant visceral yolk sac endoderm cells exhibit altered endocytic compartments, abundant autophagic vacuoles and mislocalization of endocytic markers.

Exon 5 starts from about 11.2% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 834 bp, and the size of intron 5 for 3'-loxP site insertion: 4378 bp. The size of effective cKO region: ~622 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 4 5 26 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Snx13 Homology arm cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7122bp) | A(28.12% 2003) | C(16.68% 1188) | T(36.51% 2600) | G(18.69% 1331)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr12 + 35083614 35086613 3000 browser details YourSeq 140 1460 1610 3000 96.7% chr3 + 118372880 118373377 498 browser details YourSeq 123 1169 1612 3000 77.2% chr17 + 33353603 33353920 318 browser details YourSeq 120 1477 1610 3000 95.5% chr16 - 28313926 28314511 586 browser details YourSeq 115 1376 1611 3000 84.7% chr2 + 82556316 82556519 204 browser details YourSeq 98 1279 1611 3000 95.4% chr10 - 34783453 34783807 355 browser details YourSeq 95 1512 1611 3000 98.0% chr15 + 35035394 35035506 113 browser details YourSeq 92 1512 1610 3000 97.0% chr13 + 76088448 76088559 112 browser details YourSeq 91 1512 1611 3000 96.0% chr7 - 67301036 67301148 113 browser details YourSeq 91 1512 1611 3000 96.0% chr6 + 25296087 25296199 113 browser details YourSeq 91 1512 1611 3000 96.0% chr12 + 7642002 7642114 113 browser details YourSeq 90 1512 1610 3000 96.0% chr7 - 32864494 32864605 112 browser details YourSeq 90 1512 1610 3000 96.0% chr7 - 33091390 33091501 112 browser details YourSeq 90 1512 1610 3000 96.0% chr6 - 64380799 64380910 112 browser details YourSeq 90 1520 1611 3000 99.0% chr4 - 52031915 52032006 92 browser details YourSeq 90 1520 1611 3000 99.0% chr7 + 67899728 67899819 92 browser details YourSeq 89 1520 1610 3000 99.0% chr5 - 105349759 105349849 91 browser details YourSeq 89 1512 1611 3000 95.0% chr2 - 178089036 178089148 113 browser details YourSeq 89 1520 1610 3000 99.0% chr16 - 4165214 4165304 91 browser details YourSeq 89 1512 1611 3000 95.0% chr12 - 65179929 65180041 113

Note: The 3000 bp section upstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

BLAT Search Results (down)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr12 + 35087236 35090235 3000 browser details YourSeq 755 900 1813 3000 91.8% chr3 - 54938183 55042401 104219 browser details YourSeq 678 900 1815 3000 88.8% chr15 - 61202834 61203760 927 browser details YourSeq 623 908 1806 3000 88.5% chr13 - 88437516 88438297 782 browser details YourSeq 598 1021 1816 3000 90.9% chr6 - 28023905 28024734 830 browser details YourSeq 577 1114 1806 3000 92.4% chr9 - 87629140 87630115 976 browser details YourSeq 544 1112 1815 3000 90.6% chrX - 63681243 63682246 1004 browser details YourSeq 544 1012 1813 3000 88.6% chr9 + 74581528 74582328 801 browser details YourSeq 535 1032 1806 3000 90.7% chr17 + 3915032 3915901 870 browser details YourSeq 497 941 1781 3000 88.9% chr5 - 56034032 56034720 689 browser details YourSeq 496 1106 1751 3000 91.9% chr6 - 131260214 131261016 803 browser details YourSeq 494 1195 1817 3000 90.2% chr8 + 61020391 61021386 996 browser details YourSeq 471 941 1813 3000 88.8% chr1 + 101068870 101069744 875 browser details YourSeq 417 900 1772 3000 86.4% chr2 - 142829676 142830216 541 browser details YourSeq 417 1326 1814 3000 93.0% chr3 + 68909660 68910150 491 browser details YourSeq 413 1038 1812 3000 88.9% chr10 - 130188917 130189577 661 browser details YourSeq 413 1326 1815 3000 91.8% chr7 + 105542473 105542958 486 browser details YourSeq 410 1326 1806 3000 93.3% chr15 + 91867210 91867690 481 browser details YourSeq 409 1326 1815 3000 92.4% chr4 - 110009890 110010384 495 browser details YourSeq 409 1326 1815 3000 91.9% chr15 - 13427959 13428451 493

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Snx13 sorting nexin 13 [ Mus musculus (house mouse) ] Gene ID: 217463, updated on 12-Aug-2019

Gene summary

Official Symbol Snx13 provided by MGI Official Full Name sorting nexin 13 provided by MGI Primary source MGI:MGI:2661416 See related Ensembl:ENSMUSG00000020590 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Rgs-px1; mKIAA0713 Expression Ubiquitous expression in cerebellum adult (RPKM 6.5), frontal lobe adult (RPKM 6.1) and 28 other tissues See more Orthologs human all

Genomic context

Location: 12; 12 A3 See Snx13 in Genome Data Viewer

Exon count: 26

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (35046147..35147480)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (35731861..35832149)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 7 transcripts

Gene: Snx13 ENSMUSG00000020590

Description sorting nexin 13 [Source:MGI Symbol;Acc:MGI:2661416] Gene Synonyms RGS-PX1 Location Chromosome 12: 35,047,186-35,147,479 forward strand. GRCm38:CM001005.2 About this gene This gene has 7 transcripts (splice variants), 208 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Snx13- ENSMUST00000048519.16 6231 958aa ENSMUSP00000038430.9 Protein coding CCDS56837 E9QNG6 TSL:1 201 GENCODE basic APPRIS P1

Snx13- ENSMUST00000163677.2 3681 778aa ENSMUSP00000130182.2 Protein coding - E9Q7T4 TSL:1 202 GENCODE basic

Snx13- ENSMUST00000221272.1 2027 44aa ENSMUSP00000152167.1 Nonsense mediated - A0A1Y7VKV2 TSL:1 203 decay

Snx13- ENSMUST00000222101.1 1145 No - Retained intron - - TSL:1 207 protein

Snx13- ENSMUST00000221876.1 3676 No - lncRNA - - TSL:1 206 protein

Snx13- ENSMUST00000221870.1 781 No - lncRNA - - TSL:3 205 protein

Snx13- ENSMUST00000221831.1 361 No - lncRNA - - TSL:3 204 protein

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120.29 kb Forward strand 35.04Mb 35.06Mb 35.08Mb 35.10Mb 35.12Mb 35.14Mb Genes (Comprehensive set... Snx13-201 >protein coding

Snx13-202 >protein coding Gm48137-201 >TEC

Snx13-203 >nonsense mediated decay

Snx13-204 >lncRNA Snx13-206 >lncRNA

Snx13-207 >retained intron

Gm10933-201 >TEC Snx13-205 >lncRNA

Contigs < AC140376.3 < AC122314.4 Regulatory Build

35.04Mb 35.06Mb 35.08Mb 35.10Mb 35.12Mb 35.14Mb Reverse strand 120.29 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000048519

100.29 kb Forward strand

Snx13-201 >protein coding

ENSMUSP00000038... Transmembrane heli... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily RGS domain superfamily PX domain superfamily

SMART Phox-associated domain RGS domain Phox homologous domain

Pfam Phox-associated domain RGS domain Phox homologous domain Sorting nexin, C-terminal

PROSITE profiles Phox-associated domain RGS domain Phox homologous domain

PANTHER PTHR22775

PTHR22775:SF3 Gene3D 1.20.58.1850 PX domain superfamily

CDD SNX13, RGS domain Sorting nexin-13, PX domain

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend

stop gained missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 958

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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