https://www.alphaknockout.com

Mouse Snx18 Knockout Project (CRISPR/Cas9)

Objective: To create a Snx18 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Snx18 (NCBI Reference Sequence: NM_130796 ; Ensembl: ENSMUSG00000042364 ) is located on Mouse 13. 2 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 2 (Transcript: ENSMUST00000109241). Exon 1 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note:

Exon 1 starts from the coding region. Exon 1 covers 86.4% of the coding region. The size of effective KO region: ~1594 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2

Legends Exon of mouse Snx18 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 1594 bp section of Exon 1 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 1594 bp section of Exon 1 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(1594bp) | A(18.19% 290) | C(33.81% 539) | T(16.25% 259) | G(31.74% 506)

Note: The 1594 bp section of Exon 1 is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(1594bp) | A(18.26% 291) | C(33.88% 540) | T(16.25% 259) | G(31.62% 504)

Note: The 1594 bp section of Exon 1 is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 1594 1 1594 1594 100.0% chr13 - 113616801 113618394 1594 browser details YourSeq 167 855 1188 1594 78.3% chr9 - 56925715 56926039 325 browser details YourSeq 22 1237 1261 1594 95.9% chr11 + 81082062 81082088 27 browser details YourSeq 20 837 856 1594 100.0% chr1 - 133728638 133728657 20 browser details YourSeq 20 1389 1408 1594 100.0% chr14 + 8323085 8323104 20

Note: The 1594 bp section of Exon 1 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 1594 1 1594 1594 100.0% chr13 - 113616803 113618396 1594 browser details YourSeq 167 857 1190 1594 78.3% chr9 - 56925715 56926039 325 browser details YourSeq 22 1239 1263 1594 95.9% chr11 + 81082062 81082088 27 browser details YourSeq 20 839 858 1594 100.0% chr1 - 133728638 133728657 20 browser details YourSeq 20 1391 1410 1594 100.0% chr14 + 8323085 8323104 20

Note: The 1594 bp section of Exon 1 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Snx18 18 [ Mus musculus (house mouse) ] Gene ID: 170625, updated on 12-Aug-2019

Gene summary

Official Symbol Snx18 provided by MGI Official Full Name sorting nexin 18 provided by MGI Primary source MGI:MGI:2137642 See related Ensembl:ENSMUSG00000042364 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 Snag1 Expression Ubiquitous expression in ovary adult (RPKM 28.8), bladder adult (RPKM 12.6) and 27 other tissues See more Orthologs human all

Genomic context

Location: 13; 13 D2.2 See Snx18 in Genome Data Viewer Exon count: 2

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (113592179..113618564, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (114382387..114408772, complement)

Chromosome 13 - NC_000079.6

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Transcript information: This gene has 3 transcripts

Gene: Snx18 ENSMUSG00000042364

Description sorting nexin 18 [Source:MGI Symbol;Acc:MGI:2137642] Gene Synonyms Snag1 Location Chromosome 13: 113,592,179-113,618,564 reverse strand. GRCm38:CM001006.2 About this gene This gene has 3 transcripts (splice variants), 242 orthologues, 15 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Snx18-201 ENSMUST00000109241.4 4447 615aa ENSMUSP00000104864.3 Protein coding CCDS36784 Q8C788 TSL:1 GENCODE basic APPRIS P1

Snx18-202 ENSMUST00000223993.1 3269 No protein - lncRNA - - -

Snx18-203 ENSMUST00000224883.1 549 No protein - lncRNA - - -

46.39 kb Forward strand 113.59Mb 113.60Mb 113.61Mb 113.62Mb Contigs AC168056.3 > (Comprehensive set... < Snx18-201protein coding

< Snx18-202lncRNA

< Snx18-203lncRNA

Regulatory Build

113.59Mb 113.60Mb 113.61Mb 113.62Mb Reverse strand 46.39 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000109241

< Snx18-201protein coding

Reverse strand 26.39 kb

ENSMUSP00000104... MobiDB lite Low complexity (Seg) Superfamily SH3-like domain superfamily PX domain superfamily

SMART SH3 domain Phox homologous domain

Pfam SH3 domain Sorting nexin protein, WASP-binding domain

Phox homologous domain PROSITE profiles SH3 domain Phox homologous domain

PIRSF Sorting nexin 9 family PANTHER PTHR45827:SF4

PTHR45827 Gene3D 2.30.30.40 PX domain superfamily AH/BAR domain superfamily

CDD Sorting nexin-18, SH3 domain SNX18, PX domain cd07670

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 615

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

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