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Mouse Exph5 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Exph5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Exph5 (NCBI Reference Sequence: NM_176846 ; Ensembl: ENSMUSG00000034584 ) is located on Mouse 9. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000051014). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Exph5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-93P8 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:

Exon 2 starts from about 2.04% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 35980 bp, and the size of intron 2 for 3'-loxP site insertion: 2349 bp. The size of effective cKO region: ~661 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 2 6 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Exph5 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(7161bp) | A(30.08% 2154) | C(20.65% 1479) | T(28.88% 2068) | G(20.39% 1460)

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% chr9 + 53334583 53337582 3000 browser details YourSeq 41 2215 2497 3000 78.8% chr19 + 54878858 54879129 272 browser details YourSeq 40 1955 2235 3000 95.6% chr3 + 28587625 28587955 331 browser details YourSeq 28 8 70 3000 93.8% chr19 - 12400439 12400501 63 browser details YourSeq 28 10 47 3000 96.7% chr1 + 9654613 9654651 39 browser details YourSeq 26 1955 1985 3000 82.8% chr5 + 60705236 60705264 29 browser details YourSeq 25 1955 1984 3000 82.2% chr4 - 51627331 51627358 28 browser details YourSeq 25 1295 1324 3000 82.2% chr13 + 49514839 49514866 28 browser details YourSeq 24 1953 1985 3000 88.5% chr4 + 31451001 31451032 32 browser details YourSeq 24 1955 1985 3000 84.7% chr15 + 96281725 96281753 29 browser details YourSeq 24 1953 1985 3000 88.5% chr10 + 117399820 117399851 32 browser details YourSeq 22 37 64 3000 89.3% chr16 - 97275149 97275176 28 browser details YourSeq 21 1955 1985 3000 83.9% chr18 - 34102066 34102096 31 browser details YourSeq 21 33 65 3000 81.9% chr10 + 82952109 82952141 33

Note: The 3000 bp section upstream of Exon 2 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% chr9 + 53338244 53341243 3000 browser details YourSeq 43 11 167 3000 92.0% chr11 + 114593503 114593666 164 browser details YourSeq 42 1372 1767 3000 57.4% chr14 + 24809059 24809202 144 browser details YourSeq 39 347 469 3000 95.3% chr1 - 157865284 157865432 149 browser details YourSeq 34 20 84 3000 94.8% chr2 - 117563031 117563096 66 browser details YourSeq 33 1313 1444 3000 94.8% chr17 + 8729552 8729728 177 browser details YourSeq 31 388 422 3000 94.3% chrX - 20731234 20731268 35 browser details YourSeq 31 8 82 3000 71.3% chr15 - 42709630 42710134 505 browser details YourSeq 28 22 79 3000 96.7% chr11 - 43756276 43756335 60 browser details YourSeq 28 445 472 3000 100.0% chr5 + 132715640 132715667 28 browser details YourSeq 28 1183 1231 3000 80.0% chr2 + 48517896 48517943 48 browser details YourSeq 27 1773 1799 3000 100.0% chr2 - 4384621 4384647 27 browser details YourSeq 27 21 80 3000 91.0% chr1 + 120560751 120560812 62 browser details YourSeq 25 69 169 3000 62.4% chr2 + 28179500 28179600 101 browser details YourSeq 25 1776 1804 3000 93.2% chr18 + 67376418 67376446 29 browser details YourSeq 24 545 571 3000 96.3% chr14 - 52578065 52578096 32 browser details YourSeq 24 553 581 3000 81.5% chr1 - 186065966 186065992 27 browser details YourSeq 24 445 470 3000 88.0% chr2 + 17168815 17168839 25 browser details YourSeq 23 21 49 3000 77.0% chr13 + 97571461 97571486 26 browser details YourSeq 23 446 470 3000 87.5% chr13 + 28790206 28790229 24

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Exph5 exophilin 5 [ Mus musculus (house mouse) ] Gene ID: 320051, updated on 12-Aug-2019

Gene summary

Official Symbol Exph5 provided by MGI Official Full Name exophilin 5 provided by MGI Primary source MGI:MGI:2443248 See related Ensembl:ENSMUSG00000034584 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 Slac2b; slac2-b; Kiaa0624; E030050P12; B130009M24Rik; AC079869.22gm5 Expression Biased expression in cerebellum adult (RPKM 6.3), large intestine adult (RPKM 1.7) and 10 other tissues See more Orthologs all

Genomic context

Location: 9; 9 A5.3 See Exph5 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (53301514..53381166)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (53109775..53189263)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 3 transcripts

Gene: Exph5 ENSMUSG00000034584

Description exophilin 5 [Source:MGI Symbol;Acc:MGI:2443248] Gene Synonyms AC079869.22gm5, B130009M24Rik, Slac2b, slac2-b Location Chromosome 9: 53,301,670-53,377,514 forward strand. GRCm38:CM001002.2 About this gene This gene has 3 transcripts (splice variants), 121 orthologues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Exph5-201 ENSMUST00000051014.1 5957 1960aa ENSMUSP00000062632.1 Protein coding CCDS23181 Q0VAV2 TSL:1 GENCODE basic APPRIS P1

Exph5-203 ENSMUST00000139096.1 2763 No protein - Retained intron - - TSL:1

Exph5-202 ENSMUST00000132410.1 2315 No protein - Retained intron - - TSL:1

95.84 kb Forward strand 53.30Mb 53.32Mb 53.34Mb 53.36Mb 53.38Mb (Comprehensive set... Exph5-201 >protein coding

Exph5-202 >retained intron Poglut3-201 >protein coding

Exph5-203 >retained intron

Contigs < AC160139.2 < AC079869.22 Regulatory Build

53.30Mb 53.32Mb 53.34Mb 53.36Mb 53.38Mb Reverse strand 95.84 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000051014

75.84 kb Forward strand

Exph5-201 >protein coding

ENSMUSP00000062... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) PROSITE profiles Rab-binding domain PANTHER Exophilin-5 Gene3D Zinc finger, RING/FYVE/PHD-type

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

Variant Legend missense variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1960

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

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