http://www.alphaknockout.com/

Mouse Csnk2b Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Csnk2b conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Csnk2b gene ( NCBI Reference Sequence: NM_001303476 ; Ensembl: ENSMUSG00000024387 ) is located on mouse chromosome 17. 7 exons are identified , with the ATG start codon in exon 2 and the TAA stop codon in exon 7 (Transcript: ENSMUST00000174024). 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 Csnk2b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-63A14 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 targeted mutation exhibit embryonic lethality post-implantation, growth retardation and arrest, a failure to form an inner cell mass and smaller size.

Exon 3 starts from about 9.47% 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: 588 bp, and the size of intron 3 for 3'-loxP site insertion: 1179 bp. The size of effective cKO region: ~830 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level. The expression of mouse Gpank1 and Ly6g5b may be affected by deletion of this cKO region.

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

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 5 6 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Csnk2b cKO region loxP site

Page 2 of 8 http://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(7103bp) | A(23.15% 1644) | C(25.03% 1778) | G(26.02% 1848) | T(25.81% 1833)

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.

Page 3 of 8 http://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% chr17 - 35120107 35123106 3000 browser details YourSeq 346 1 1652 3000 89.6% chr13 - 17949578 17949959 382 browser details YourSeq 136 597 854 3000 86.0% chr8 - 11045538 11045693 156 browser details YourSeq 136 586 736 3000 95.4% chr5 - 143534566 143534733 168 browser details YourSeq 133 584 731 3000 95.3% chr10 - 83267596 83267778 183 browser details YourSeq 133 595 759 3000 89.2% chr7 + 116320056 116320206 151 browser details YourSeq 132 583 736 3000 92.2% chr7 - 123072853 123073005 153 browser details YourSeq 132 593 756 3000 92.0% chr14 + 53110199 53110360 162 browser details YourSeq 129 593 731 3000 96.5% chr17 + 17459038 17459176 139 browser details YourSeq 127 593 736 3000 94.5% chr15 - 98147351 98147495 145 browser details YourSeq 127 585 736 3000 91.7% chr8 + 126941551 126941700 150 browser details YourSeq 126 594 736 3000 92.1% chr13 - 30530268 30530407 140 browser details YourSeq 126 593 736 3000 93.0% chr12 - 45642819 45642961 143 browser details YourSeq 126 596 731 3000 96.4% chr18 + 34100676 34100811 136 browser details YourSeq 126 592 736 3000 91.7% chr13 + 39848753 39848895 143 browser details YourSeq 125 595 737 3000 93.8% chr12 - 109714365 109714507 143 browser details YourSeq 125 595 731 3000 95.7% chr11 - 62686184 62686320 137 browser details YourSeq 124 596 736 3000 92.1% chr2 - 76985533 76985671 139 browser details YourSeq 124 596 729 3000 96.3% chr1 - 181124086 181124219 134 browser details YourSeq 124 591 736 3000 93.1% chr2 + 171071888 171072376 489

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% chr17 - 35116504 35119503 3000 browser details YourSeq 312 2163 2689 3000 90.1% chr11 + 88163900 88164274 375 browser details YourSeq 309 2105 2690 3000 91.7% chr15 + 8510614 8511181 568 browser details YourSeq 306 2163 2691 3000 90.1% chr15 - 76580451 76580830 380 browser details YourSeq 303 2166 2692 3000 90.8% chr5 + 65585603 65585982 380 browser details YourSeq 289 2176 2691 3000 89.1% chr7 - 12999955 13000319 365 browser details YourSeq 285 2177 2690 3000 94.2% chr14 + 31241996 31242511 516 browser details YourSeq 278 2162 2690 3000 90.3% chr18 - 56604606 56604961 356 browser details YourSeq 278 2188 2691 3000 91.7% chr1 + 164024582 164025082 501 browser details YourSeq 273 2188 2690 3000 89.3% chr1 - 59624431 59624774 344 browser details YourSeq 272 2197 2691 3000 94.5% chr5 - 147296547 147297099 553 browser details YourSeq 272 2178 2690 3000 88.8% chr2 - 144246768 144247122 355 browser details YourSeq 265 2197 2692 3000 89.1% chr2 + 181563710 181564047 338 browser details YourSeq 249 2197 2691 3000 92.7% chr17 + 23799731 23800211 481 browser details YourSeq 237 2257 2693 3000 90.4% chr17 + 28611043 28611342 300 browser details YourSeq 233 2195 2690 3000 97.6% chr16 - 22128449 22129048 600 browser details YourSeq 225 2210 2692 3000 91.1% chr3 + 90116081 90116502 422 browser details YourSeq 221 2249 2694 3000 95.9% chr1 - 74535711 74536331 621 browser details YourSeq 219 2254 2693 3000 94.0% chr11 - 101567345 101567841 497 browser details YourSeq 217 2162 2689 3000 89.7% chr4 - 59197786 59198031 246

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 http://www.alphaknockout.com/ Gene and protein information: Csnk2b casein kinase 2, beta polypeptide [ Mus musculus (house mouse) ] Gene ID: 13001, updated on 11-Sep-2019

Gene summary

Official Symbol Csnk2b provided by MGI Official Full Name casein kinase 2, beta polypeptide provided by MGI Primary source MGI:MGI:88548 See related Ensembl:ENSMUSG00000024387 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as CK II beta Summary This gene encodes the beta subunit of the casein kinase 2 enzyme, which is a heterotetramer comprised of alpha and/or Expression alpha-prime catalytic subunits and two regulatory beta subunits. Casein kinase 2 is involved in the regulation of several cellular processes including gene expression, protein synthesis and cell proliferation. Knockout of this gene in mice leads to embryonic lethality. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, Jan 2015] Orthologs Broad expression in testis adult (RPKM 540.1), duodenum adult (RPKM 75.4) and 23 other tissues See more human all

Genomic context

Location: 17 B1; 17 18.59 cM See Csnk2b in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (35116195..35122053, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (35253140..35258392, complement)

Chromosome 17 - NC_000083.6

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

Transcript information: This gene has 10 transcripts

Gene: Csnk2b ENSMUSG00000024387

Description casein kinase 2, beta polypeptide [Source:MGI Symbol;Acc:MGI:88548] Location Chromosome 17: 35,116,196-35,122,053 reverse strand. GRCm38:CM001010.2 About this gene This gene has 10 transcripts (splice variants), 216 orthologues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Csnk2b-201 ENSMUST00000025246.12 961 215aa ENSMUSP00000025246.6 Protein coding CCDS28684 P67871 TSL:1 GENCODE basic APPRIS P1

Csnk2b-203 ENSMUST00000173114.7 917 215aa ENSMUSP00000134218.1 Protein coding CCDS28684 P67871 TSL:1 GENCODE basic APPRIS P1

Csnk2b-207 ENSMUST00000174024.7 883 257aa ENSMUSP00000134673.1 Protein coding CCDS79541 G3UZX4 TSL:5 GENCODE basic

Csnk2b-202 ENSMUST00000172765.8 828 206aa ENSMUSP00000134523.2 Protein coding - G3UZJ5 CDS 3' incomplete TSL:2

Csnk2b-209 ENSMUST00000174779.7 632 164aa ENSMUSP00000133684.1 Protein coding - G3UXG7 TSL:2 GENCODE basic

Csnk2b-206 ENSMUST00000173915.2 592 58aa ENSMUSP00000133425.1 Protein coding - G3UWU5 CDS 3' incomplete TSL:3

Csnk2b-205 ENSMUST00000173633.2 555 180aa ENSMUSP00000133826.2 Protein coding - G3UXU2 CDS 5' incomplete TSL:5

Csnk2b-208 ENSMUST00000174306.7 402 122aa ENSMUSP00000134413.1 Protein coding - G3UZA4 CDS 3' incomplete TSL:5

Csnk2b-204 ENSMUST00000173189.1 590 No protein - Retained intron - - TSL:2

Csnk2b-210 ENSMUST00000174855.1 405 No protein - Retained intron - - TSL:5

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

25.86 kb Forward strand 35.11Mb 35.12Mb 35.13Mb Genes Ly6g5c-201 >protein coding Gpank1-204 >protein coding D17H6S53E-202 >retained intron (Comprehensive set...

Gpank1-203 >protein coding D17H6S53E-201 >protein coding

Gpank1-202 >protein coding

Gpank1-205 >processed transcript

Gpank1-201 >protein coding

Contigs AC087117.9 > CR974444.18 > Genes < Ly6g5b-202protein codin

< Ly6g5b-201protein codin

< Csnk2b-203protein coding

< Csnk2b-201protein coding

< Csnk2b-209protein coding < Csnk2b-210retained intron

< Csnk2b-207protein coding

< Csnk2b-205protein cod

< Csnk2b-208protein coding

Regulatory Build

35.11Mb 35.12Mb 35.13Mb Reverse strand 25.86 kb

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

Regulation Legend CTCF Enhancer Promoter Promoter Flank

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

Transcript: ENSMUST00000174024

< Csnk2b-207protein coding

Reverse strand 4.97 kb

ENSMUSP00000134... Low complexity (Seg) Superfamily Casein kinase II subunit beta-like SMART Casein kinase II, regulatory subunit Prints Casein kinase II, regulatory subunit Pfam Casein kinase II, regulatory subunit PANTHER Casein kinase II, regulatory subunit Gene3D Casein kinase II, regulatory subunit, N-terminal 2.20.25.20

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

Variant Legend stop gained frameshift variant missense variant synonymous variant

Scale bar 0 40 80 120 160 200 257

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

Page 8 of 8