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Mouse Ush2a Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ush2a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ush2a gene (NCBI Reference Sequence: NM_021408 ; Ensembl: ENSMUSG00000026609 ) is located on Mouse chromosome 1. 71 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 71 (Transcript: ENSMUST00000060479). Exon 6~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ush2a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-153E16 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 knock-out allele display progressive retinal photoreceptor degeneration along with significantly reduced a- and b-wave amplitudes, and a moderate but nonprogressive high-frequency hearing loss associated with widespread loss of outer hair cells in the basal turn of the cochlea.

Exon 6 starts from about 7.29% of the coding region. The knockout of Exon 6~7 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 872 bp, and the size of intron 7 for 3'-loxP site insertion: 1100 bp. The size of effective cKO region: ~1359 bp. The cKO 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 5 6 7 8 71 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ush2a 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(7859bp) | A(29.71% 2335) | C(21.45% 1686) | T(29.63% 2329) | G(19.2% 1509)

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% chr1 + 188354607 188357606 3000 browser details YourSeq 186 1073 1434 3000 91.2% chr7 - 40714817 40715204 388 browser details YourSeq 159 1140 1437 3000 91.5% chr1 - 159757553 159758060 508 browser details YourSeq 158 1098 1436 3000 90.0% chr8 - 120932893 120933321 429 browser details YourSeq 155 1066 1433 3000 92.4% chr8 - 27073847 27074319 473 browser details YourSeq 154 1070 1383 3000 92.9% chr1 - 190182815 190415514 232700 browser details YourSeq 151 1073 1434 3000 93.2% chr5 + 113417476 113417971 496 browser details YourSeq 150 1065 1409 3000 87.4% chr1 - 192671016 192671332 317 browser details YourSeq 138 1070 1371 3000 88.9% chr10 - 10966869 10967164 296 browser details YourSeq 137 1066 1413 3000 94.9% chr1 - 159757406 159757775 370 browser details YourSeq 134 1072 1439 3000 92.5% chr10 - 40127636 40321378 193743 browser details YourSeq 133 1095 1433 3000 78.9% chr10 - 19282037 19282253 217 browser details YourSeq 132 1075 1409 3000 83.2% chr1 + 42345513 42345747 235 browser details YourSeq 131 1070 1437 3000 82.6% chr14 + 11617834 11618069 236 browser details YourSeq 130 1079 1441 3000 80.4% chr1 + 76575939 76576162 224 browser details YourSeq 130 1070 1434 3000 80.9% chr1 + 7118825 7119029 205 browser details YourSeq 128 1124 1343 3000 91.1% chr1 + 190637581 190638050 470 browser details YourSeq 127 1129 1419 3000 88.8% chr12 - 106104247 106111413 7167 browser details YourSeq 125 1074 1337 3000 91.8% chr5 + 139025803 139026302 500 browser details YourSeq 125 1075 1384 3000 91.3% chr1 + 192210146 192210541 396

Note: The 3000 bp section upstream of Exon 6 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% chr1 + 188358966 188361965 3000 browser details YourSeq 344 2471 3000 3000 87.7% chr11 - 93933420 94088546 155127 browser details YourSeq 342 2469 2989 3000 91.6% chr5 - 20117700 20768277 650578 browser details YourSeq 321 2500 3000 3000 89.9% chr7 + 93032361 93033051 691 browser details YourSeq 315 2471 3000 3000 86.4% chr2 - 97917739 97918233 495 browser details YourSeq 309 2469 3000 3000 89.8% chr16 + 9490539 9491166 628 browser details YourSeq 285 2587 3000 3000 87.5% chr13 + 108784131 108784758 628 browser details YourSeq 219 2480 3000 3000 85.7% chr6 - 9762286 9762723 438 browser details YourSeq 217 2708 2999 3000 89.3% chr7 - 17600259 17695713 95455 browser details YourSeq 214 2747 3000 3000 92.9% chr6 + 115118960 115119216 257 browser details YourSeq 213 2720 3000 3000 88.5% chrX + 72470403 72470686 284 browser details YourSeq 210 2720 3000 3000 89.6% chr18 + 23145308 23145592 285 browser details YourSeq 207 2745 3000 3000 91.0% chr14 + 77784939 77785199 261 browser details YourSeq 207 2720 3000 3000 89.4% chr11 + 38929851 38930131 281 browser details YourSeq 206 2720 2991 3000 91.6% chr5 + 69034650 69034927 278 browser details YourSeq 203 2745 3000 3000 90.2% chr12 - 88612643 88612903 261 browser details YourSeq 203 2720 2983 3000 90.0% chrX + 72974047 72974308 262 browser details YourSeq 200 2749 2989 3000 93.5% chr11 - 110076594 110076838 245 browser details YourSeq 199 2751 3000 3000 90.4% chr8 - 32479681 32479935 255 browser details YourSeq 198 2745 3000 3000 89.0% chr9 - 118275610 118275866 257

Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ush2a usherin [ Mus musculus (house mouse) ] Gene ID: 22283, updated on 10-Oct-2019

Gene summary

Official Symbol Ush2a provided by MGI Official Full Name usherin provided by MGI Primary source MGI:MGI:1341292 See related Ensembl:ENSMUSG00000026609 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 Gm676; Gm983; Ushrn; Mush2a; A930011D15Rik; A930037M10Rik Expression Biased expression in testis adult (RPKM 1.3) and ovary adult (RPKM 0.0) See more Orthologs human all

Genomic context

Location: 1 H6; 1 92.29 cM See Ush2a in Genome Data Viewer

Exon count: 73

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (188262838..188965497)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (190086717..190788920)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 5 transcripts

Gene: Ush2a ENSMUSG00000026609

Description usherin [Source:MGI Symbol;Acc:MGI:1341292] Gene Synonyms A930011D15Rik, A930037M10Rik, LOC269160, LOC381317, MUSH2A, Ush2a, Ushrn Location Chromosome 1: 188,262,023-188,965,041 forward strand. GRCm38:CM000994.2 About this gene This gene has 5 transcripts (splice variants), 182 orthologues, 26 paralogues, is a member of 2 Ensembl protein families and is associated with 9 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ush2a-201 ENSMUST00000060479.13 15695 5193aa ENSMUSP00000050454.7 Protein coding CCDS15607 Q2QI47 TSL:1 GENCODE basic APPRIS P1

Ush2a-203 ENSMUST00000127077.1 750 237aa ENSMUSP00000121033.1 Protein coding - F6TQ19 CDS 5' incomplete TSL:5

Ush2a-202 ENSMUST00000124358.1 5126 No protein - Retained intron - - TSL:1

Ush2a-204 ENSMUST00000142159.1 2282 No protein - Retained intron - - TSL:1

Ush2a-205 ENSMUST00000142189.7 1236 No protein - Retained intron - - TSL:1

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723.02 kb Forward strand

Genes (Comprehensive set... Gm38064-201 >lncRNA Ush2a-204 >retained intron Gm25269-201 >snRNA Ush2a-203 >protein coding

Ush2a-205 >retained intron

Ush2a-202 >retained intron

Ush2a-201 >protein coding

Contigs < AC165230.12 < AC121892.3 < AC121799.5 < AC135863.4 AC129312.6 > Genes < Gm25095-201snoRNA < Kctd3-204nonsense mediated decay (Comprehensive set...

< Kctd3-202retained intron

< Kctd3-209retained intron

< Kctd3-207retained intron

< Kctd3-201protein coding

< Kctd3-208protein coding

< Kctd3-203protein coding

Regulatory Build

Reverse strand 723.02 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000060479

702.01 kb Forward strand

Ush2a-201 >protein coding

ENSMUSP00000050... Transmembrane heli... MobiDB lite Low complexity (Seg) Superfamily Concanavalin A-like lectin/glucanase domain superfamily

SSF57196 Fibronectin type III superfamily SMART Laminin EGF domain Laminin G domain

LamG-like jellyroll fold Fibronectin type III

Laminin, N-terminal Prints PR00011 Pfam Laminin, N-terminal Laminin G domain

PF13385 Laminin EGF domain Laminin G domain

Fibronectin type III PROSITE profiles Laminin, N-terminal Fibronectin type III

Laminin EGF domain Laminin G domain PROSITE patterns Laminin EGF domain PANTHER PTHR10574

Usherin Gene3D 2.60.120.200

Laminin, N-terminal domain superfamily

2.10.25.10 Immunoglobulin-like fold CDD cd00055 Fibronectin type III

cd00110

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

Variant Legend stop gained frameshift variant inframe insertion missense variant splice region variant synonymous variant

Scale bar 0 600 1200 1800 2400 3000 3600 4200 5193

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

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