https://www.alphaknockout.com Mouse Snw1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Snw1 conditional knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Snw1 (NCBI Reference Sequence: NM_025507 ; Ensembl: ENSMUSG00000021039 ) is located on Mouse 12. 14 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 14 (Transcript: ENSMUST00000021428). 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 Snw1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-425D9 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 0.93% 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: 3484 bp, and the size of intron 2 for 3'-loxP site insertion: 3898 bp. The size of effective cKO region: ~2160 bp. The cKO region does not have any other known gene.

Page 1 of 8 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Snw1 Homology arm cKO region loxP site

Page 2 of 8 https://www.alphaknockout.com

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(7154bp) | A(27.69% 1981) | C(20.6% 1474) | G(22.31% 1596) | T(29.4% 2103)

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 8 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% chr12 - 87469014 87472013 3000 browser details YourSeq 441 2413 3000 3000 94.6% chr9 + 108195837 108196553 717 browser details YourSeq 433 2410 3000 3000 91.2% chr5 + 36724712 36725224 513 browser details YourSeq 421 2081 2860 3000 93.1% chr12 + 53505751 53506530 780 browser details YourSeq 415 2410 2967 3000 93.9% chr1 - 165732507 165733191 685 browser details YourSeq 412 2410 2869 3000 95.8% chr7 - 6462698 6463173 476 browser details YourSeq 411 2410 2872 3000 96.2% chr8 - 104399078 104399548 471 browser details YourSeq 411 2410 2869 3000 95.8% chr3 - 99489432 99489894 463 browser details YourSeq 408 2410 2872 3000 95.2% chr9 + 94342554 94343026 473 browser details YourSeq 407 2410 2955 3000 92.0% chrX - 74549959 74550458 500 browser details YourSeq 407 2410 2870 3000 94.8% chrX + 140865149 140865626 478 browser details YourSeq 406 2410 2981 3000 93.5% chr9 - 119480006 119480787 782 browser details YourSeq 406 2410 2870 3000 95.0% chr11 - 35645053 35645522 470 browser details YourSeq 405 2423 2978 3000 91.0% chr5 - 130486360 130486876 517 browser details YourSeq 404 2410 2870 3000 95.0% chr3 - 152527246 152527717 472 browser details YourSeq 404 2410 2872 3000 94.7% chr10 - 94070104 94070585 482 browser details YourSeq 404 2411 2870 3000 95.2% chr6 + 128148017 128148498 482 browser details YourSeq 404 2410 2868 3000 95.1% chr16 + 6546373 6546835 463 browser details YourSeq 404 2410 2865 3000 95.5% chr11 + 28905402 28905871 470 browser details YourSeq 403 2412 2870 3000 94.3% chr16 + 12385307 12385769 463

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% chr12 - 87465360 87468359 3000 browser details YourSeq 193 2527 2750 3000 93.4% chr4 - 129179369 129356925 177557 browser details YourSeq 191 2503 2733 3000 92.5% chr10 - 81339270 81339878 609 browser details YourSeq 188 2362 2733 3000 93.2% chr9 - 121307688 121308070 383 browser details YourSeq 186 2538 2772 3000 91.9% chr1 - 80400308 80400808 501 browser details YourSeq 180 2516 2733 3000 93.4% chr16 - 90982336 90982555 220 browser details YourSeq 177 2509 2737 3000 89.7% chr1 - 193533120 193533346 227 browser details YourSeq 174 2362 2737 3000 93.6% chr16 + 13956815 13957193 379 browser details YourSeq 173 2528 2733 3000 92.2% chr5 + 106796933 106797139 207 browser details YourSeq 171 2512 2725 3000 90.9% chr18 + 56451562 56451779 218 browser details YourSeq 171 2528 2725 3000 93.9% chr10 + 80346999 80347199 201 browser details YourSeq 170 2533 2731 3000 93.0% chr6 - 41357056 41357258 203 browser details YourSeq 170 2546 2766 3000 92.5% chr17 + 15614055 15614355 301 browser details YourSeq 169 2538 2733 3000 94.3% chr7 - 80658609 80658809 201 browser details YourSeq 169 2533 2725 3000 93.8% chr1 - 101579171 101579363 193 browser details YourSeq 168 2533 2733 3000 92.1% chr3 + 96748099 96748300 202 browser details YourSeq 167 2525 2727 3000 90.4% chr7 - 92751949 92752148 200 browser details YourSeq 167 2532 2731 3000 92.5% chr6 + 38546986 38547195 210 browser details YourSeq 166 2528 2725 3000 92.0% chr11 - 107516301 107516498 198 browser details YourSeq 166 2362 2733 3000 95.2% chr9 + 110317510 110318107 598

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

Page 4 of 8 https://www.alphaknockout.com Gene and information: Snw1 SNW domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 66354, updated on 12-Aug-2019

Gene summary

Official Symbol Snw1 provided by MGI Official Full Name SNW domain containing 1 provided by MGI Primary source MGI:MGI:1913604 See related Ensembl:ENSMUSG00000021039 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 SKIP; Skiip; NCoA-62; AW048543; 2310008B08Rik Expression Broad expression in CNS E11.5 (RPKM 29.9), bladder adult (RPKM 19.2) and 23 other tissues See more Orthologs human all

Genomic context

Location: 12; 12 D2 See Snw1 in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (87449910..87472299, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (88790860..88813249, complement)

Chromosome 12 - NC_000078.6

Page 5 of 8 https://www.alphaknockout.com

Transcript information: This gene has 3 transcripts

Gene: Snw1 ENSMUSG00000021039

Description SNW domain containing 1 [Source:MGI Symbol;Acc:MGI:1913604] Gene Synonyms 2310008B08Rik, NCoA-62, SKIP, SNW1, Skiip Location Chromosome 12: 87,449,075-87,472,274 reverse strand. GRCm38:CM001005.2 About this gene This gene has 3 transcripts (splice variants), 208 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Snw1-201 ENSMUST00000021428.8 2232 536aa ENSMUSP00000021428.7 Protein coding CCDS49121 A0A0B4J1E2 TSL:1 GENCODE basic APPRIS P1

Snw1-202 ENSMUST00000222579.1 5083 No protein - Retained intron - - TSL:NA

Snw1-203 ENSMUST00000223119.1 479 No protein - Retained intron - - TSL:NA

Page 6 of 8 https://www.alphaknockout.com

43.20 kb Forward strand 87.44Mb 87.45Mb 87.46Mb 87.47Mb 87.48Mb Slirp-204 >protein coding Gm18983-201 >processed pseudogene (Comprehensive set...

Slirp-202 >protein coding

Slirp-201 >protein coding

Slirp-203 >protein coding

Contigs CT030249.7 > < AC126554.3 Genes (Comprehensive set... < Alkbh1-209protein coding < Snw1-202retained intron < Snw1-203retained intron

< Alkbh1-208nonsense mediated decay < Snw1-201protein coding

< Alkbh1-203nonsense mediated decay

< Alkbh1-207nonsense mediated decay

< Alkbh1-202nonsense mediated decay

< Alkbh1-206lncRNA

< Alkbh1-210nonsense mediated decay

< Alkbh1-205retained intron

< Alkbh1-201retained intron

< Alkbh1-212protein coding

Regulatory Build

87.44Mb 87.45Mb 87.46Mb 87.47Mb 87.48Mb Reverse strand 43.20 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene pseudogene processed transcript

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000021428

< Snw1-201protein coding

Reverse strand 22.36 kb

ENSMUSP00000021... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam SKI-interacting protein SKIP, SNW domain

PANTHER SKI-interacting protein, SKIP

PTHR12096:SF0

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 536

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

Page 8 of 8