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Mouse Cyth1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cyth1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cyth1 gene (NCBI Reference Sequence: NM_011180 ; Ensembl: ENSMUSG00000017132 ) is located on Mouse chromosome 11. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000106305). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cyth1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-370L14 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 gene trap allele exhibit normal brain morphology and long term potentiation. Mice homozygous for a knock-out allele exhibit decreased myelin sheath thickness due to hypomyelination.

Exon 3 starts from about 8.88% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1153 bp, and the size of intron 3 for 3'-loxP site insertion: 6570 bp. The size of effective cKO region: ~565 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 3 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cyth1 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(7065bp) | A(24.46% 1728) | C(24.06% 1700) | T(27.3% 1929) | G(24.18% 1708)

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% chr11 - 118192712 118195711 3000 browser details YourSeq 121 2804 2993 3000 90.7% chr6 - 28288724 28289076 353 browser details YourSeq 121 2796 2931 3000 94.9% chr15 + 81701531 81701678 148 browser details YourSeq 115 2798 2932 3000 92.6% chr11 - 86482727 86482861 135 browser details YourSeq 114 2796 2932 3000 92.0% chr11 + 32111504 32111649 146 browser details YourSeq 113 2796 2932 3000 93.9% chrX + 132531256 132531405 150 browser details YourSeq 112 2800 2931 3000 90.0% chr2 - 167047094 167047222 129 browser details YourSeq 112 2798 2931 3000 91.8% chr11 - 82762521 82762654 134 browser details YourSeq 112 2796 2931 3000 91.2% chr9 + 57308258 57308393 136 browser details YourSeq 110 2804 2933 3000 92.4% chr19 + 19917178 19917307 130 browser details YourSeq 110 2798 2931 3000 91.1% chr15 + 37284003 37284136 134 browser details YourSeq 109 2810 2932 3000 92.7% chr5 - 139519205 139519326 122 browser details YourSeq 107 2804 2934 3000 90.9% chr9 - 121249161 121249291 131 browser details YourSeq 107 2800 2928 3000 91.5% chr2 - 69676794 69676922 129 browser details YourSeq 107 2798 2931 3000 90.3% chr10 + 70162215 70162350 136 browser details YourSeq 106 2809 2928 3000 94.2% chr5 - 28233443 28233562 120 browser details YourSeq 106 2809 2928 3000 94.2% chr6 + 119742070 119742189 120 browser details YourSeq 106 2809 2932 3000 91.1% chr10 + 7810841 7810963 123 browser details YourSeq 105 2800 2930 3000 90.1% chrX - 128364462 128364592 131 browser details YourSeq 105 2804 2930 3000 91.4% chr5 - 111246601 111246727 127

Note: The 3000 bp section upstream of Exon 3 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% chr11 - 118189147 118192146 3000 browser details YourSeq 368 656 1055 3000 96.0% chr11 - 118191252 118191651 400 browser details YourSeq 368 496 895 3000 96.0% chr11 - 118191092 118191491 400 browser details YourSeq 222 816 1055 3000 96.3% chr11 - 118191412 118191651 240 browser details YourSeq 222 496 735 3000 96.3% chr11 - 118191092 118191331 240 browser details YourSeq 202 211 2519 3000 91.3% chr13 + 19762560 19996131 233572 browser details YourSeq 193 1728 2517 3000 92.2% chr11 + 46316585 46386134 69550 browser details YourSeq 174 496 683 3000 96.3% chr11 - 118191104 118191291 188 browser details YourSeq 162 2361 2996 3000 84.0% chr4 + 129386156 129386366 211 browser details YourSeq 147 2354 2526 3000 95.8% chrX - 166603120 166603315 196 browser details YourSeq 145 2357 2518 3000 95.7% chr15 - 102613978 102614144 167 browser details YourSeq 145 2357 2518 3000 95.1% chr15 + 82828957 82829119 163 browser details YourSeq 145 2359 2523 3000 91.4% chr13 + 113331205 113331366 162 browser details YourSeq 145 2357 2522 3000 94.6% chr1 + 60418516 60418688 173 browser details YourSeq 144 2363 2527 3000 94.6% chr15 - 61575595 61575892 298 browser details YourSeq 144 2354 2523 3000 91.2% chr14 - 31848753 31848917 165 browser details YourSeq 143 2358 2527 3000 93.5% chr1 - 192656864 192657166 303 browser details YourSeq 143 2354 2527 3000 93.3% chr2 + 30059688 30060188 501 browser details YourSeq 142 2359 2518 3000 92.5% chr19 - 45992537 45992694 158 browser details YourSeq 142 2358 2523 3000 94.4% chr8 + 59506608 59506779 172

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cyth1 cytohesin 1 [ Mus musculus (house mouse) ] Gene ID: 19157, updated on 24-Oct-2019

Gene summary

Official Symbol Cyth1 provided by MGI Official Full Name cytohesin 1 provided by MGI Primary source MGI:MGI:1334257 See related Ensembl:ENSMUSG00000017132 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 CLM1; CTH-1; CYTIP; Pscd1 Expression Ubiquitous expression in cerebellum adult (RPKM 19.7), frontal lobe adult (RPKM 16.5) and 27 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 E2 See Cyth1 in Genome Data Viewer

Exon count: 18

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (118164166..118248616, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (118025480..118109906, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 7 transcripts

Gene: Cyth1 ENSMUSG00000017132

Description cytohesin 1 [Source:MGI Symbol;Acc:MGI:1334257] Gene Synonyms CLM1, CTH-1, Pscd1 Location Chromosome 11: 118,132,019-118,248,592 reverse strand. GRCm38:CM001004.2 About this gene This gene has 7 transcripts (splice variants), 259 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

Cyth1- ENSMUST00000106305.8 3179 398aa ENSMUSP00000101912.2 Protein CCDS48996 Q9QX11 TSL:1 204 coding GENCODE basic APPRIS ALT1

Cyth1- ENSMUST00000017276.13 3135 397aa ENSMUSP00000017276.7 Protein CCDS48997 Q8K3E8 TSL:1 201 coding Q9QX11 GENCODE basic APPRIS ALT1

Cyth1- ENSMUST00000106302.8 3119 400aa ENSMUSP00000101909.2 Protein CCDS48995 Q3TZ02 TSL:1 203 coding GENCODE basic APPRIS P4

Cyth1- ENSMUST00000100181.10 1592 460aa ENSMUSP00000097756.4 Protein - A2A517 CDS 5' 202 coding incomplete TSL:1

Cyth1- ENSMUST00000151165.1 363 99aa ENSMUSP00000114792.1 Protein - B1AQE4 CDS 3' 207 coding incomplete TSL:2

Cyth1- ENSMUST00000141243.7 545 No - lncRNA - - TSL:3 206 protein

Cyth1- ENSMUST00000131115.1 355 No - lncRNA - - TSL:3 205 protein

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136.57 kb Forward strand 118.14Mb 118.16Mb 118.18Mb 118.20Mb 118.22Mb 118.24Mb Contigs AL591204.14 > AL591109.8 > AL591404.4 > Genes (Comprehensive set... < Dnah17-203protein coding < Cyth1-204protein coding

< Dnah17-202protein coding < Cyth1-203protein coding

< Dnah17-204protein coding < Cyth1-201protein coding

< Cyth1-202protein coding

< Gm24060-201snRNA < Gm11737-201processed pseudogene

< Cyth1-206lncRNA

< Cyth1-205lncRNA

< Cyth1-207protein coding

Regulatory Build

118.14Mb 118.16Mb 118.18Mb 118.20Mb 118.22Mb 118.24Mb Reverse strand 136.57 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

pseudogene RNA gene

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Transcript: ENSMUST00000106305

< Cyth1-204protein coding

Reverse strand 84.43 kb

ENSMUSP00000101... 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:SF340

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

synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 398

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

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