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Mouse Wbp11 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Wbp11 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Wbp11 gene (NCBI Reference Sequence: NM_021714 ; Ensembl: ENSMUSG00000030216 ) is located on Mouse chromosome 6. 12 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000116514). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Wbp11 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-93A18 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 2 starts from about 100% of the coding region. The knockout of Exon 2~4 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1910 bp, and the size of intron 4 for 3'-loxP site insertion: 1339 bp. The size of effective cKO region: ~2235 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 2 3 4 5 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Wbp11 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(8735bp) | A(27.52% 2404) | C(20.47% 1788) | T(27.84% 2432) | G(24.17% 2111)

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% chr6 - 136826315 136829314 3000 browser details YourSeq 350 40 510 3000 89.8% chr16 + 35979411 35979895 485 browser details YourSeq 335 56 510 3000 89.4% chr11 - 35371436 35371904 469 browser details YourSeq 326 74 510 3000 88.0% chr13 - 97555850 97556311 462 browser details YourSeq 323 56 510 3000 88.5% chr10 + 12969336 12969816 481 browser details YourSeq 321 69 510 3000 89.7% chr1 - 40798796 40799259 464 browser details YourSeq 321 39 510 3000 87.8% chr7 + 40648656 40649161 506 browser details YourSeq 319 56 506 3000 90.4% chr8 + 64912594 64913061 468 browser details YourSeq 317 56 506 3000 89.4% chr6 - 92076047 92076523 477 browser details YourSeq 317 38 510 3000 90.0% chr4 + 140080160 140080664 505 browser details YourSeq 313 78 510 3000 89.1% chr11 - 23910222 23910673 452 browser details YourSeq 312 74 510 3000 89.3% chr15 - 97560304 97560805 502 browser details YourSeq 311 89 510 3000 89.1% chr13 + 35148764 35149206 443 browser details YourSeq 310 56 510 3000 90.7% chr1 - 132172257 132172721 465 browser details YourSeq 310 74 510 3000 91.0% chr6 + 90886366 90886838 473 browser details YourSeq 309 96 510 3000 89.4% chr17 + 63751953 63752388 436 browser details YourSeq 306 74 510 3000 89.5% chr7 - 135202833 135203282 450 browser details YourSeq 302 56 507 3000 90.0% chr5 - 101684029 101684509 481 browser details YourSeq 302 33 506 3000 88.5% chr18 - 61188618 61189088 471 browser details YourSeq 302 56 510 3000 87.8% chr9 + 109829168 109829806 639

Note: The 3000 bp section upstream of Exon 2 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% chr6 - 136821080 136824079 3000 browser details YourSeq 446 62 548 3000 96.0% chr11 + 18201227 18201700 474 browser details YourSeq 440 62 522 3000 98.1% chrX - 153376154 153376682 529 browser details YourSeq 440 62 525 3000 97.9% chr13 - 27481941 27482413 473 browser details YourSeq 440 62 525 3000 97.7% chr4 + 135259877 135260340 464 browser details YourSeq 439 62 528 3000 96.3% chr4 - 100931909 100932369 461 browser details YourSeq 439 62 522 3000 97.0% chr13 - 93649534 93649990 457 browser details YourSeq 439 62 518 3000 98.3% chr8 + 68754711 68755341 631 browser details YourSeq 439 62 527 3000 97.5% chr12 + 6157186 6157659 474 browser details YourSeq 438 62 518 3000 98.1% chr18 + 17170472 17170930 459 browser details YourSeq 437 62 522 3000 97.0% chr13 - 110036115 110036572 458 browser details YourSeq 437 62 526 3000 96.5% chr11 - 56564596 56565053 458 browser details YourSeq 437 62 521 3000 97.0% chr1 + 194988592 194989047 456 browser details YourSeq 436 62 522 3000 97.9% chr7 - 73862720 73863194 475 browser details YourSeq 436 62 523 3000 97.4% chr10 + 31123340 31123802 463 browser details YourSeq 435 62 523 3000 97.4% chrX - 36732130 36732766 637 browser details YourSeq 435 62 518 3000 97.2% chr9 - 6896084 6896537 454 browser details YourSeq 435 48 514 3000 97.4% chr5 - 83983178 83983643 466 browser details YourSeq 435 62 517 3000 98.1% chr4 - 96408226 96408685 460 browser details YourSeq 435 62 518 3000 97.9% chr2 - 84935417 84936326 910

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Wbp11 WW domain binding protein 11 [ Mus musculus (house mouse) ] Gene ID: 60321, updated on 12-Aug-2019

Gene summary

Official Symbol Wbp11 provided by MGI Official Full Name WW domain binding protein 11 provided by MGI Primary source MGI:MGI:1891823 See related Ensembl:ENSMUSG00000030216 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 Npwbp; SIPP1; D6Wsu113e; 2510026P17Rik Expression Ubiquitous expression in testis adult (RPKM 80.0), CNS E11.5 (RPKM 24.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 6 G1; 6 66.72 cM See Wbp11 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (136813654..136828216, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (136762175..136776737, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 7 transcripts

Gene: Wbp11 ENSMUSG00000030216

Description WW domain binding protein 11 [Source:MGI Symbol;Acc:MGI:1891823] Gene Synonyms 2510026P17Rik, D6Wsu113e, Npwbp, SIPP1 Location Chromosome 6: 136,813,654-136,828,233 reverse strand. GRCm38:CM000999.2 About this gene This gene has 7 transcripts (splice variants), 11 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Wbp11- ENSMUST00000116514.3 2739 641aa ENSMUSP00000112213.1 Protein coding CCDS20655 Q923D5 TSL:1 201 GENCODE basic APPRIS P1

Wbp11- ENSMUST00000204129.1 427 143aa ENSMUSP00000144977.1 Protein coding - A0A0N4SV69 CDS 5' and 3' 206 incomplete TSL:1

Wbp11- ENSMUST00000146348.3 312 32aa ENSMUSP00000145155.1 Protein coding - A0A0N4SVL7 CDS 3' 204 incomplete TSL:5

Wbp11- ENSMUST00000204272.2 905 194aa ENSMUSP00000145501.1 Nonsense mediated - A0A0N4SWF7 TSL:5 207 decay

Wbp11- ENSMUST00000151333.1 2339 No - Retained intron - - TSL:1 205 protein

Wbp11- ENSMUST00000141598.2 779 No - Retained intron - - TSL:5 203 protein

Wbp11- ENSMUST00000129078.1 536 No - Retained intron - - TSL:2 202 protein

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34.58 kb Forward strand 136.81Mb 136.82Mb 136.83Mb Genes Gm43969-201 >TEC BC049715-205 >retained intron (Comprehensive set...

H2afj-201 >protein coding BC049715-204 >protein coding

H2afj-202 >protein coding BC049715-201 >protein coding

BC049715-203 >protein coding

Contigs < AC122804.4

Genes (Comprehensive set... < Hist4h4-201protein codin

< Wbp11-206protein coding < Wbp11-203retained intron < Smco3-203protein coding

< Wbp11-207nonsense mediated decay < Smco3-202protein coding

< Wbp11-205retained intron

< Wbp11-202retained intron

< Wbp11-204protein coding

Regulatory Build

136.81Mb 136.82Mb 136.83Mb Reverse strand 34.58 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000116514

< Wbp11-201protein coding

Reverse strand 14.58 kb

ENSMUSP00000112... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam WW domain binding protein 11 PANTHER PTHR13361:SF2

WW domain binding protein 11

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

Variant Legend stop gained missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 641

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

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