https://www.alphaknockout.com

Mouse Wipi2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Wipi2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Wipi2 gene (NCBI Reference Sequence: NM_178398 ; Ensembl: ENSMUSG00000029578 ) is located on Mouse chromosome 5. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000036872). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Wipi2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-324G18 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 5.62% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 8369 bp, and the size of intron 2 for 3'-loxP site insertion: 17563 bp. The size of effective cKO region: ~583 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Wipi2 Homology arm cKO region loxP site

Page 2 of 7 https://www.alphaknockout.com

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(7083bp) | A(24.75% 1753) | C(21.9% 1551) | T(28.29% 2004) | G(25.06% 1775)

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.

Page 3 of 7 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr5 + 142634953 142637952 3000 browser details YourSeq 83 2360 2490 3000 90.3% chr6 + 72377218 72377364 147 browser details YourSeq 80 2364 2465 3000 90.9% chr13 + 55184035 55184137 103 browser details YourSeq 78 2364 2899 3000 71.5% chr1 - 127682561 127682974 414 browser details YourSeq 78 2345 2465 3000 82.3% chr5 + 31128993 31129109 117 browser details YourSeq 77 2364 2465 3000 88.3% chr11 - 118143687 118143789 103 browser details YourSeq 74 2329 2465 3000 87.0% chr11 - 5304753 5304885 133 browser details YourSeq 73 2344 2465 3000 83.7% chr5 - 73035326 73035442 117 browser details YourSeq 73 2364 2465 3000 86.8% chr11 - 20709131 20709234 104 browser details YourSeq 73 2364 2465 3000 89.3% chr5 + 127808917 127809019 103 browser details YourSeq 72 2369 2464 3000 90.0% chr15 - 83380509 83380604 96 browser details YourSeq 72 2345 2465 3000 83.7% chr2 + 140725074 140725189 116 browser details YourSeq 71 2364 2465 3000 85.3% chr5 - 129716979 129717081 103 browser details YourSeq 71 2364 2465 3000 85.2% chr1 - 138463600 138463701 102 browser details YourSeq 71 2366 2465 3000 89.2% chr12 + 80680650 80680750 101 browser details YourSeq 70 2364 2463 3000 86.0% chr13 - 34673915 34674016 102 browser details YourSeq 69 2364 2465 3000 84.4% chr8 - 24700675 24700777 103 browser details YourSeq 69 2364 2461 3000 86.5% chr5 - 138833071 138833168 98 browser details YourSeq 67 2364 2465 3000 87.0% chr17 - 26750016 26750117 102 browser details YourSeq 67 2353 2465 3000 84.1% chr1 + 23753947 23754056 110

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% chr5 + 142638536 142641535 3000 browser details YourSeq 355 1405 2107 3000 86.0% chrX - 17725357 17726163 807 browser details YourSeq 348 1444 2107 3000 87.7% chr11 - 9494953 9495696 744 browser details YourSeq 333 1380 2106 3000 87.9% chr11 - 48691279 48692039 761 browser details YourSeq 294 1516 2107 3000 87.2% chrX - 102568241 102568887 647 browser details YourSeq 283 1471 2087 3000 88.2% chr5 + 135135954 135136622 669 browser details YourSeq 266 1675 2361 3000 90.9% chr8 + 45857397 45858299 903 browser details YourSeq 259 1527 2072 3000 87.4% chr10 + 21374335 21374886 552 browser details YourSeq 248 1600 2107 3000 83.6% chr4 + 16466977 16467469 493 browser details YourSeq 238 1568 2084 3000 87.7% chr11 + 22650781 22651305 525 browser details YourSeq 232 1558 2107 3000 89.2% chr2 + 50549889 50550478 590 browser details YourSeq 204 1518 2002 3000 88.6% chr6 + 67557663 67558179 517 browser details YourSeq 198 1568 1995 3000 86.1% chr12 + 9918960 9919424 465 browser details YourSeq 195 1601 2055 3000 86.6% chr6 + 63047223 63047676 454 browser details YourSeq 186 1659 2107 3000 87.8% chr7 - 101166896 101167331 436 browser details YourSeq 185 1455 1922 3000 85.3% chr13 - 11682341 11682848 508 browser details YourSeq 161 1568 1912 3000 83.9% chr8 + 62995777 62996122 346 browser details YourSeq 160 1651 2124 3000 87.1% chr4 - 112249302 112249804 503 browser details YourSeq 155 1468 1757 3000 84.0% chr4 - 110044820 110045117 298 browser details YourSeq 155 1544 1922 3000 85.5% chr1 - 117487294 117487699 406

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 7 https://www.alphaknockout.com

Gene and protein information: Wipi2 WD repeat domain, phosphoinositide interacting 2 [ Mus musculus (house mouse) ] Gene ID: 74781, updated on 12-Aug-2019

Gene summary

Official Symbol Wipi2 provided by MGI Official Full Name WD repeat domain, phosphoinositide interacting 2 provided by MGI Primary source MGI:MGI:1923831 See related Ensembl:ENSMUSG00000029578 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as 1110018O08Rik; 2510001I10Rik Expression Ubiquitous expression in ovary adult (RPKM 25.2), adrenal adult (RPKM 22.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 G2 See Wipi2 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (142629533..142669672)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (143105538..143145326)

Chromosome 5 - NC_000071.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 5 transcripts

Gene: Wipi2 ENSMUSG00000029578

Description WD repeat domain, phosphoinositide interacting 2 [Source:MGI Symbol;Acc:MGI:1923831] Gene Synonyms 1110018O08Rik, 2510001I10Rik Location Chromosome 5: 142,627,698-142,670,588 forward strand. GRCm38:CM000998.2 About this gene This gene has 5 transcripts (splice variants), 210 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Wipi2-201 ENSMUST00000036872.15 5171 445aa ENSMUSP00000045201.9 Protein coding CCDS19828 Q80W47 TSL:1 GENCODE basic

Wipi2-202 ENSMUST00000110778.1 1959 425aa ENSMUSP00000106405.1 Protein coding - D3YWK1 TSL:5 GENCODE basic APPRIS P1

Wipi2-204 ENSMUST00000153936.1 3244 No protein - Retained intron - - TSL:1

Wipi2-205 ENSMUST00000197864.1 2793 No protein - Retained intron - - TSL:NA

Wipi2-203 ENSMUST00000143980.1 949 No protein - Retained intron - - TSL:2

62.89 kb Forward strand

Genes (Comprehensive set... Wipi2-205 >retained intron Wipi2-203 >retained intron

Wipi2-201 >protein coding

Wipi2-204 >retained intron

Wipi2-202 >protein coding

Contigs AC158310.3 > < AC105987.21 Genes < Gm31274-201processed pseudogene (Comprehensive set...

Regulatory Build

Reverse strand 62.89 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript pseudogene

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000036872

41.05 kb Forward strand

Wipi2-201 >protein coding

ENSMUSP00000045... Superfamily WD40-repeat-containing domain superfamily SMART WD40 repeat PANTHER WD repeat domain phosphoinositide-interacting protein 2

PTHR11227 Gene3D WD40/YVTN repeat-like-containing domain superfamily

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 445

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

Page 7 of 7