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Mouse Ssr2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ssr2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ssr2 gene (NCBI Reference Sequence: NM_025448 ; Ensembl: ENSMUSG00000041355 ) is located on Mouse chromosome 3. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000035785). 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 Ssr2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-174J9 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 3 starts from about 46.45% 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: 2704 bp, and the size of intron 3 for 3'-loxP site insertion: 2979 bp. The size of effective cKO region: ~649 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 3 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ssr2 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7109bp) | A(22.91% 1629) | C(23.35% 1660) | T(31.59% 2246) | G(22.14% 1574)

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% chr3 + 88580528 88583527 3000 browser details YourSeq 329 1049 1698 3000 88.1% chr2 - 168714908 168715434 527 browser details YourSeq 296 1116 1698 3000 93.9% chr1 + 136694073 136755709 61637 browser details YourSeq 291 1008 1702 3000 92.7% chr11 + 120211272 120601529 390258 browser details YourSeq 282 1171 1702 3000 94.1% chr19 - 7010882 7011476 595 browser details YourSeq 280 1069 1662 3000 87.8% chr7 - 16292377 16292962 586 browser details YourSeq 280 1170 1698 3000 89.8% chr13 + 9404342 9404682 341 browser details YourSeq 279 1170 1698 3000 90.6% chr13 - 64130499 64130879 381 browser details YourSeq 270 1169 1690 3000 89.7% chr2 + 126654729 126655050 322 browser details YourSeq 268 1002 1378 3000 92.0% chr12 - 111635714 111636336 623 browser details YourSeq 267 1172 1702 3000 95.0% chr4 - 127001201 127001743 543 browser details YourSeq 259 1169 1690 3000 94.3% chr1 - 7046165 7046688 524 browser details YourSeq 258 1169 1698 3000 91.2% chr9 - 107677820 107678260 441 browser details YourSeq 243 1169 1684 3000 88.2% chr7 - 19930973 19931297 325 browser details YourSeq 243 1171 1689 3000 85.3% chr2 + 30087897 30088214 318 browser details YourSeq 239 1049 1358 3000 93.8% chr11 - 78312121 78312602 482 browser details YourSeq 238 1170 1686 3000 90.7% chr9 + 54584390 54584900 511 browser details YourSeq 236 1050 1358 3000 93.1% chr18 - 81002859 81003452 594 browser details YourSeq 236 1049 1350 3000 91.6% chr11 + 120601424 120601745 322 browser details YourSeq 233 1051 1358 3000 92.7% chr9 + 21526981 21527506 526

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% chr3 + 88584137 88587136 3000 browser details YourSeq 1184 32 1457 3000 91.9% chr12 + 9144677 9146104 1428 browser details YourSeq 1163 29 1457 3000 91.7% chr1 - 181027955 181029383 1429 browser details YourSeq 1163 30 1457 3000 91.3% chr14 + 68160598 68162025 1428 browser details YourSeq 1158 30 1457 3000 90.8% chr13 - 103692984 103694405 1422 browser details YourSeq 1156 26 1457 3000 90.5% chr1 + 13757951 13759378 1428 browser details YourSeq 1154 31 1457 3000 91.6% chr15 + 59576299 59577745 1447 browser details YourSeq 1150 33 1454 3000 91.1% chr3 - 58824281 58825700 1420 browser details YourSeq 1150 26 1457 3000 90.3% chr15 + 34591702 34593126 1425 browser details YourSeq 1148 33 1460 3000 91.7% chr18 - 14560405 14561832 1428 browser details YourSeq 1148 33 1449 3000 92.3% chr12 + 52613398 52614823 1426 browser details YourSeq 1147 29 1455 3000 90.2% chrX + 166249378 166250801 1424 browser details YourSeq 1146 31 1457 3000 90.9% chr16 - 34522767 34524197 1431 browser details YourSeq 1145 31 1457 3000 91.4% chr14 - 66632522 66633965 1444 browser details YourSeq 1143 32 1570 3000 91.1% chr9 - 20420020 20421872 1853 browser details YourSeq 1143 30 1449 3000 90.9% chr11 - 5658352 5659775 1424 browser details YourSeq 1141 31 1457 3000 90.5% chr18 - 21443260 21444683 1424 browser details YourSeq 1141 31 1457 3000 91.1% chr3 + 157260876 157513530 252655 browser details YourSeq 1138 31 1457 3000 90.6% chr2 - 84327780 84329205 1426 browser details YourSeq 1137 29 1457 3000 90.9% chr17 + 55754020 55755451 1432

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: Ssr2 signal sequence receptor, beta [ Mus musculus (house mouse) ] Gene ID: 66256, updated on 24-Oct-2019

Gene summary

Official Symbol Ssr2 provided by MGI Official Full Name signal sequence receptor, beta provided by MGI Primary source MGI:MGI:1913506 See related Ensembl:ENSMUSG00000041355 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 TLAP; TRAPB; AI315033; AU020133; SSR-beta; TRAPbeta; 1500032E05Rik Expression Ubiquitous expression in placenta adult (RPKM 192.7), large intestine adult (RPKM 147.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 F1 See Ssr2 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (88579608..88588420)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (88383593..88392335)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 7 transcripts

Gene: Ssr2 ENSMUSG00000041355

Description signal sequence receptor, beta [Source:MGI Symbol;Acc:MGI:1913506] Gene Synonyms TRAPbeta Location Chromosome 3: 88,575,876-88,588,419 forward strand. GRCm38:CM000996.2 About this gene This gene has 7 transcripts (splice variants), 203 orthologues, is a member of 1 Ensembl protein family and is associated with 43 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ssr2-201 ENSMUST00000035785.8 1189 183aa ENSMUSP00000045456.7 Protein coding CCDS17480 Q9CPW5 TSL:1 GENCODE basic APPRIS P1

Ssr2-207 ENSMUST00000195014.5 1059 183aa ENSMUSP00000141441.1 Protein coding CCDS17480 Q9CPW5 TSL:1 GENCODE basic APPRIS P1

Ssr2-202 ENSMUST00000192495.5 620 185aa ENSMUSP00000141922.1 Protein coding - A0A0A6YXB7 TSL:5 GENCODE basic

Ssr2-205 ENSMUST00000193934.5 569 164aa ENSMUSP00000141325.1 Protein coding - A0A0A6YVZ1 TSL:3 GENCODE basic

Ssr2-203 ENSMUST00000192688.5 560 117aa ENSMUSP00000141471.1 Protein coding - A0A0A6YWA8 CDS 3' incomplete TSL:3

Ssr2-204 ENSMUST00000193069.1 495 34aa ENSMUSP00000141389.1 Protein coding - A0A0A6YW45 CDS 3' incomplete TSL:3

Ssr2-206 ENSMUST00000194604.1 494 32aa ENSMUSP00000141591.1 Protein coding - A0A0A6YWL0 CDS 3' incomplete TSL:3

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32.54 kb Forward strand 88.57Mb 88.58Mb 88.59Mb Genes (Comprehensive set... Ubqln4-201 >protein coding Ssr2-203 >protein coding

Ssr2-204 >protein coding

Ssr2-206 >protein coding

Ssr2-207 >protein coding

Ssr2-205 >protein coding

Ssr2-202 >protein coding

Ssr2-201 >protein coding

Contigs AC145168.5 >

Genes < Rtraf-ps-201processed pseudogene (Comprehensive set...

Regulatory Build

88.57Mb 88.58Mb 88.59Mb Reverse strand 32.54 kb

Regulation Legend

CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

pseudogene

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Transcript: ENSMUST00000035785

8.75 kb Forward strand

Ssr2-201 >protein coding

ENSMUSP00000045... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Pfam PF05753 PIRSF Translocon-associated protein subunit beta PANTHER PTHR12861

PTHR12861:SF3

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 183

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

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