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Mouse Cyth2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cyth2 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cyth2 gene ( NCBI Reference Sequence: NM_001112701 ; Ensembl: ENSMUSG00000003269 ) is located on mouse chromosome 7. 12 exons are identified , with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000107729). Exon 4~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the mouse Cyth2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-36B17 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: Mice homozygous for a conditional allele activated in Schwann cell exhibit reduced sciatic nerve myelin sheath thickness.

The knockout of Exon 4~8 will result in frameshift of the gene, and covers 47.95% of the coding region. The size of intron 3 for 5'-loxP site insertion: 548 bp, and the size of intron 8 for 3'-loxP site insertion: 1677 bp. The size of effective cKO region: ~2818 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3' 10

1 2 3 4 5 6 7 8 9 11 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Cyth2 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(9172bp) | A(22.09% 2026) | C(26.3% 2412) | G(28.58% 2621) | T(23.04% 2113)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr7 - 45812549 45815548 3000 browser details YourSeq 117 262 405 3000 90.9% chr2 + 25179223 25179378 156 browser details YourSeq 113 273 428 3000 90.8% chr18 + 36309764 36309999 236 browser details YourSeq 112 267 406 3000 92.5% chr13 - 23507075 23507228 154 browser details YourSeq 108 273 405 3000 91.0% chr7 - 45799609 45799754 146 browser details YourSeq 106 266 406 3000 88.5% chr10 - 117404153 117404326 174 browser details YourSeq 105 276 404 3000 91.5% chr4 + 116080963 116081108 146 browser details YourSeq 104 273 406 3000 90.0% chr1 - 68262272 68262414 143 browser details YourSeq 103 274 405 3000 90.0% chr13 - 29872461 29872608 148 browser details YourSeq 101 273 394 3000 91.8% chr15 - 81818728 81818862 135 browser details YourSeq 101 274 405 3000 88.6% chr12 - 85002883 85003027 145 browser details YourSeq 100 276 394 3000 92.4% chr1 - 79720474 79720605 132 browser details YourSeq 100 276 394 3000 92.4% chr1 - 79727916 79728047 132 browser details YourSeq 98 282 405 3000 90.2% chr16 + 18497638 18497776 139 browser details YourSeq 96 276 404 3000 89.4% chr1 - 39355562 39355710 149 browser details YourSeq 95 273 394 3000 89.3% chr11 - 16452325 16452462 138 browser details YourSeq 95 276 405 3000 88.0% chr6 + 11676536 11676680 145 browser details YourSeq 94 273 406 3000 88.0% chr12 - 80804734 80804888 155 browser details YourSeq 94 273 405 3000 90.6% chr11 - 82865748 82865919 172 browser details YourSeq 94 273 394 3000 93.6% chr5 + 21905415 21905550 136

Note: The 3000 bp section upstream of Exon 4 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% chr7 - 45806877 45809876 3000 browser details YourSeq 136 1436 1803 3000 81.8% chr5 + 143707092 143707408 317 browser details YourSeq 112 1434 1706 3000 85.9% chr11 - 118170821 118177290 6470 browser details YourSeq 62 1421 1494 3000 91.9% chr15 + 78618369 78618442 74 browser details YourSeq 24 424 454 3000 84.7% chr3 - 86857894 86857922 29

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 http://beta.alphaknockout.cyagen.net Gene and protein information: Cyth2 cytohesin 2 [ Mus musculus (house mouse) ] Gene ID: 19158, updated on 15-Aug-2019

Gene summary

Official Symbol Cyth2 provided by MGI Official Full Name cytohesin 2 provided by MGI Primary source MGI:MGI:1334255 See related Ensembl:ENSMUSG00000003269 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 ARNO; CLM2; Pscd2 Expression Ubiquitous expression in CNS E11.5 (RPKM 32.8), CNS E14 (RPKM 32.4) and 28 other tissues See more Orthologs human all

Genomic context

Location: 7; 7 B3 See Cyth2 in Genome Data Viewer

Exon count: 13

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (45806637..45814322, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (53062007..53069686, complement)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 9 transcripts

Gene: Cyth2 ENSMUSG00000003269

Description cytohesin 2 [Source:MGI Symbol;Acc:MGI:1334255] Gene Synonyms ARNO, CLM2, Pscd2 Location Chromosome 7: 45,806,637-45,814,581 reverse strand. GRCm38:CM001000.2 About this gene This gene has 9 transcripts (splice variants), 184 orthologues, 15 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cyth2- ENSMUST00000107729.9 2519 399aa ENSMUSP00000103357.1 Protein coding CCDS52249 Q99KH2 TSL:1 202 GENCODE basic APPRIS P2

Cyth2- ENSMUST00000056820.12 2527 400aa ENSMUSP00000051423.6 Protein coding - P63034 TSL:1 201 GENCODE basic APPRIS ALT1

Cyth2- ENSMUST00000211783.1 1983 383aa ENSMUSP00000147706.1 Protein coding - A0A1B0GRX7 TSL:5 208 GENCODE basic

Cyth2- ENSMUST00000211263.1 689 166aa ENSMUSP00000147587.1 Protein coding - A0A1B0GRM7 CDS 3' 207 incomplete TSL:5

Cyth2- ENSMUST00000223361.1 604 202aa ENSMUSP00000152189.1 Protein coding - A0A1Y7VIW9 CDS 5' and 3' 209 incomplete TSL:5

Cyth2- ENSMUST00000210898.1 538 140aa ENSMUSP00000147275.1 Protein coding - A0A1B0GQW2 CDS 5' 206 incomplete TSL:5

Cyth2- ENSMUST00000209245.1 1512 292aa ENSMUSP00000147457.1 Nonsense mediated - A0A1B0GRB8 TSL:5 203 decay

Cyth2- ENSMUST00000210853.2 1510 283aa ENSMUSP00000147451.2 Nonsense mediated - A0A1B0GRB2 TSL:5 205 decay

Cyth2- ENSMUST00000210137.1 691 124aa ENSMUSP00000147723.1 Nonsense mediated - A0A1B0GRZ2 CDS 5' 204 decay incomplete TSL:5

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27.95 kb Forward strand 45.80Mb 45.81Mb 45.82Mb Genes Lmtk3-204 >protein coding Gm45444-201 >TEC (Comprehensive set...

Lmtk3-201 >protein coding

Lmtk3-202 >protein coding

Lmtk3-209 >protein coding

Contigs AC167242.4 > < AC149053.3 Genes < Cyth2-201protein coding < Kcnj14-201protein coding (Comprehensive set...

< Cyth2-202protein coding < Kcnj14-202protein coding

< Cyth2-204nonsense mediated decay

< Cyth2-203nonsense mediated decay

< Cyth2-205nonsense mediated decay

< Cyth2-208protein coding

< Cyth2-209protein coding

< Cyth2-207protein coding

< Cyth2-206protein coding

Regulatory Build

45.80Mb 45.81Mb 45.82Mb Reverse strand 27.95 kb

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

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Transcript: ENSMUST00000107729

< Cyth2-202protein coding

Reverse strand 7.67 kb

ENSMUSP00000103... Coiled-coils (Ncoils) Superfamily Sec7 domain superfamily SSF50729

SMART Sec7 domain Pleckstrin homology domain

Pfam Sec7 domain Pleckstrin homology domain

PROSITE profiles Sec7 domain Pleckstrin homology domain

PANTHER PTHR10663:SF343

PTHR10663 Gene3D 1.10.220.20 Sec7, C-terminal domain superfamily PH-like domain superfamily

CDD Sec7 domain cd01252

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

Variant Legend

missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 399

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

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