Mouse Stau1 Conditional Knockout Project (CRISPR/Cas9)

https://www.alphaknockout.com Mouse Stau1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Stau1 conditional knockout mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Stau1 gene (NCBI Reference Sequence: NM_001109906 ; Ensembl: ENSMUSG00000039536 ) is located on mouse chromosome 2. 12 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000109238). 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 Stau1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-41G23 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 targeted allele exhibit hypoactivity and impaired dendrite outgrowth and spine formation.

Exon 3 starts from about 5.45% 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: 984 bp, and the size of intron 3 for 3'-loxP site insertion: 3307 bp. The size of effective cKO region: ~1040 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 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Stau1 Homology arm cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

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

Summary: Full Length(7166bp) | A(23.88% 1711) | C(20.81% 1491) | T(30.16% 2161) | G(25.16% 1803)

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% chr2 - 166963845 166966844 3000 browser details YourSeq 468 42 1419 3000 91.1% chr10 + 76072886 76210161 137276 browser details YourSeq 388 452 1384 3000 91.5% chr4 - 135316578 136012364 695787 browser details YourSeq 250 117 1025 3000 87.4% chr4 + 124787867 124788589 723 browser details YourSeq 238 1071 1413 3000 88.1% chr5 + 76853057 76853383 327 browser details YourSeq 237 1072 1419 3000 87.4% chr4 + 137066163 137066515 353 browser details YourSeq 235 1073 1407 3000 84.5% chr4 - 86763211 86763542 332 browser details YourSeq 233 1072 1411 3000 89.8% chr17 - 30722181 30722533 353 browser details YourSeq 227 1074 1411 3000 86.0% chr16 + 18533485 18533822 338 browser details YourSeq 226 1070 1404 3000 84.3% chrX + 157544270 157544602 333 browser details YourSeq 222 1073 1420 3000 89.1% chr11 + 48777724 48778068 345 browser details YourSeq 219 1073 1410 3000 86.1% chr18 + 64900882 64901217 336 browser details YourSeq 219 37 639 3000 89.9% chr11 + 104324292 104324694 403 browser details YourSeq 218 42 639 3000 88.0% chr11 - 72298148 72298591 444 browser details YourSeq 216 167 653 3000 96.2% chr17 + 65954856 65955493 638 browser details YourSeq 215 1073 1407 3000 91.6% chr19 - 41805027 41805364 338 browser details YourSeq 214 316 1117 3000 93.6% chr10 + 60250937 60251834 898 browser details YourSeq 212 1098 1407 3000 83.0% chr7 - 79484683 79484984 302 browser details YourSeq 212 1073 1382 3000 90.2% chr6 - 52710631 52710942 312 browser details YourSeq 212 1073 1410 3000 86.0% chr17 + 32299583 32299923 341

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% chr2 - 166960179 166963178 3000 browser details YourSeq 252 889 1173 3000 94.4% chr4 - 116867380 116867665 286 browser details YourSeq 248 875 1172 3000 91.5% chrX + 66689243 66689537 295 browser details YourSeq 231 874 1172 3000 91.4% chr1 - 75459891 75460222 332 browser details YourSeq 227 888 1165 3000 91.1% chr8 - 122528585 122528860 276 browser details YourSeq 226 874 1171 3000 89.6% chr12 + 73496979 73497268 290 browser details YourSeq 220 902 1172 3000 91.7% chr8 + 94048835 94049122 288 browser details YourSeq 217 892 1170 3000 90.1% chr11 - 84994965 84995291 327 browser details YourSeq 204 893 1123 3000 94.4% chr5 - 136557962 136558245 284 browser details YourSeq 203 927 1176 3000 91.5% chr13 - 37744246 37744496 251 browser details YourSeq 203 1715 1924 3000 99.1% chr11 - 97311912 97312127 216 browser details YourSeq 203 1696 1915 3000 99.1% chr5 + 30213240 30213871 632 browser details YourSeq 201 1713 1924 3000 97.7% chr18 + 4319410 4319624 215 browser details YourSeq 200 1713 1921 3000 99.1% chr11 - 7811400 7811641 242 browser details YourSeq 199 1713 1921 3000 96.6% chr2 - 30201491 30201696 206 browser details YourSeq 197 1721 1932 3000 98.1% chr18 - 60662459 60662671 213 browser details YourSeq 197 1721 1921 3000 99.1% chr8 + 107334722 107334922 201 browser details YourSeq 195 1715 1923 3000 95.6% chr6 - 5051999 5052203 205 browser details YourSeq 195 1713 1915 3000 97.6% chr13 + 55587135 55587336 202 browser details YourSeq 195 1721 1921 3000 98.6% chr10 + 45481995 45482195 201

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

Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Stau1 staufen double-stranded RNA binding protein 1 [ Mus musculus (house mouse) ] Gene ID: 20853, updated on 7-Jan-2018

Gene summary

Official Symbol Stau1 provided by MGI Official Full Name staufen double-stranded RNA binding protein 1 provided by MGI Primary source MGI:MGI:1338864 See related Ensembl:ENSMUSG00000039536 Vega:OTTMUSG00000001134 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 Stau; C85792; AW549911; 5830401L18Rik Expression Ubiquitous expression in CNS E14 (RPKM 16.8), adrenal adult (RPKM 16.1) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 H3 See Stau1 in Genome Data Viewer Map Viewer Exon count: 12

Annotation release Status Assembly Chr Location

106 current GRCm38.p4 (GCF_000001635.24) 2 NC_000068.7 (166947549..166996299, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (166773641..166821778, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 11 transcripts

Gene: Stau1 ENSMUSG00000039536

Description staufen double-stranded RNA binding protein 1 [Source:MGI Symbol;Acc:MGI:1338864] Gene Synonyms 5830401L18Rik Location Chromosome 2: 166,947,549-166,996,299 reverse strand. GRCm38:CM000995.2 About this gene This gene has 11 transcripts (splice variants), 205 orthologues, 14 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Stau1- ENSMUST00000109235.7 3569 489aa ENSMUSP00000104858.1 Protein coding CCDS50800 Q9DBE7 TSL:1 202 GENCODE basic APPRIS ALT2

Stau1- ENSMUST00000109238.8 2976 495aa ENSMUSP00000104861.2 Protein coding CCDS50801 A2A5S3 TSL:1 204 GENCODE basic APPRIS ALT2

Stau1- ENSMUST00000109236.8 2860 487aa ENSMUSP00000104859.2 Protein coding CCDS17093 Q9Z108 TSL:1 203 GENCODE basic APPRIS P3

Stau1- ENSMUST00000049412.11 2840 485aa ENSMUSP00000042626.5 Protein coding CCDS71197 A2A5R8 TSL:1 201 GENCODE basic APPRIS ALT2

Stau1- ENSMUST00000184390.1 1513 436aa ENSMUSP00000139039.1 Nonsense mediated - V9GX87 TSL:1 211 decay

Stau1- ENSMUST00000142481.7 3573 No - Retained intron - - TSL:2 208 protein

Stau1- ENSMUST00000134664.1 862 No - Retained intron - - TSL:3 207 protein

Stau1- ENSMUST00000154506.1 826 No - Retained intron - - TSL:3 210 protein

Stau1- ENSMUST00000130790.1 788 No - Retained intron - - TSL:5 206 protein

Stau1- ENSMUST00000149454.1 774 No - Retained intron - - TSL:2 209 protein

Stau1- ENSMUST00000130104.1 341 No - lncRNA - - TSL:3 205 protein

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68.75 kb Forward strand 166.94Mb 166.96Mb 166.98Mb 167.00Mb Genes Cse1l-212 >lncRNA (Comprehensive set...

Cse1l-201 >protein coding

Cse1l-211 >nonsense mediated decay

Cse1l-210 >protein coding

Cse1l-206 >protein coding

Cse1l-205 >retained intron

Cse1l-208 >nonsense mediated decay

Cse1l-207 >retained intron

Cse1l-202 >retained intron

Gm17096-201 >lncRNA

Contigs AL591711.22 >

Genes < Stau1-202protein coding (Comprehensive set...

< Stau1-204protein coding

< Stau1-201protein coding

< Stau1-208retained intron < Stau1-205lncRNA

< Stau1-203protein coding

< Stau1-209retained intron

< Stau1-211nonsense mediated decay

< Stau1-207retained intron

< Stau1-210retained intron

< Stau1-206retained intron

Regulatory Build

166.94Mb 166.96Mb 166.98Mb 167.00Mb Reverse strand 68.75 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000109238

< Stau1-204protein coding

Reverse strand 48.12 kb

ENSMUSP00000104... MobiDB lite Low complexity (Seg) Superfamily SSF54768 SMART Double-stranded RNA-binding domain Pfam Double-stranded RNA-binding domain Staufen, C-terminal

PROSITE profiles Double-stranded RNA-binding domain PANTHER PTHR46054

PTHR46054:SF2 Gene3D 3.30.160.20 CDD Double-stranded RNA-binding domain

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

Variant Legend

missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 495

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

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