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Mouse Csnk1d Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Csnk1d conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Csnk1d (NCBI Reference Sequence: NM_139059 ; Ensembl: ENSMUSG00000025162 ) is located on Mouse 11. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000018274). 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 Csnk1d gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-446J17 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: Homozygous null mice die 1.5 to 5 days after birth and fetal weight is significantly smaller than controls.

Exon 3 starts from about 15.1% 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: 9263 bp, and the size of intron 3 for 3'-loxP site insertion: 626 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 4 5 6 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Csnk1d 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. 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(7149bp) | A(24.66% 1763) | C(21.39% 1529) | T(30.24% 2162) | G(23.71% 1695)

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% chr11 - 120974220 120977219 3000 browser details YourSeq 138 1641 1932 3000 89.4% chr1 + 190521003 190521360 358 browser details YourSeq 123 1626 1926 3000 85.0% chr3 + 101361845 101362173 329 browser details YourSeq 123 1632 1926 3000 86.3% chr10 + 63253074 63253433 360 browser details YourSeq 111 1685 1926 3000 91.8% chr13 - 52556465 52556763 299 browser details YourSeq 111 1703 1926 3000 86.4% chr2 + 157735429 157735709 281 browser details YourSeq 106 1685 1926 3000 90.3% chr5 + 133800756 133801000 245 browser details YourSeq 105 1630 1932 3000 89.5% chrX - 17628757 17629097 341 browser details YourSeq 105 1607 1934 3000 89.6% chr17 - 43848362 43848885 524 browser details YourSeq 103 1658 1922 3000 84.3% chr1 - 72798165 72798470 306 browser details YourSeq 102 1658 1932 3000 83.7% chr5 - 142826269 142826607 339 browser details YourSeq 101 1646 1918 3000 91.9% chr1 + 91796121 91796449 329 browser details YourSeq 100 1673 1932 3000 87.5% chr5 - 115791808 115792098 291 browser details YourSeq 100 1634 1778 3000 86.0% chr1 - 71783983 71784135 153 browser details YourSeq 95 1641 1778 3000 88.1% chr7 + 116447591 116447731 141 browser details YourSeq 95 1649 1782 3000 88.1% chr6 + 117680022 117680160 139 browser details YourSeq 94 1705 1932 3000 84.2% chr4 - 118605017 118605288 272 browser details YourSeq 93 1639 1782 3000 83.1% chr10 + 60240379 60240525 147 browser details YourSeq 91 1641 1783 3000 82.9% chr9 + 123414112 123414256 145 browser details YourSeq 90 1634 1783 3000 88.9% chr9 - 116566185 116566339 155

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% chr11 - 120970571 120973570 3000 browser details YourSeq 291 413 2003 3000 87.3% chr15 - 79424841 79426451 1611 browser details YourSeq 65 477 611 3000 74.7% chr10 + 80638500 80638634 135 browser details YourSeq 50 2666 2783 3000 85.8% chr5 - 34596257 34596378 122 browser details YourSeq 50 2701 2772 3000 89.1% chr5 + 76003396 76003491 96 browser details YourSeq 47 2631 2766 3000 85.1% chr11 + 53642119 53642409 291 browser details YourSeq 46 2245 2311 3000 96.0% chr6 - 122675810 122676270 461 browser details YourSeq 46 2241 2315 3000 92.8% chr13 + 63699976 63789223 89248 browser details YourSeq 42 2677 2750 3000 83.9% chr12 - 88040256 88040343 88 browser details YourSeq 40 2710 2789 3000 80.0% chr11 + 120582906 120582984 79 browser details YourSeq 38 2247 2365 3000 97.5% chr6 + 113961129 113961247 119 browser details YourSeq 37 2666 2791 3000 91.2% chr13 - 7540453 7540582 130 browser details YourSeq 37 2717 2783 3000 78.5% chr11 - 96653766 96653834 69 browser details YourSeq 36 2245 2296 3000 84.7% chr12 - 80408466 80408517 52 browser details YourSeq 34 2243 2294 3000 82.7% chr14 + 76253345 76253396 52 browser details YourSeq 34 2713 2752 3000 92.5% chr14 + 47601426 47601465 40 browser details YourSeq 33 2730 2783 3000 82.4% chr11 - 77336216 77336271 56 browser details YourSeq 33 2732 2783 3000 83.7% chr10 - 76869555 76869608 54 browser details YourSeq 33 2244 2296 3000 81.2% chr10 - 60241969 60242021 53 browser details YourSeq 33 2770 2820 3000 82.4% chr13 + 49737278 49737328 51

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 information: Csnk1d casein 1, delta [ Mus musculus (house mouse) ] Gene ID: 104318, updated on 10-Oct-2019

Gene summary

Official Symbol Csnk1d provided by MGI Official Full Name , delta provided by MGI Primary source MGI:MGI:1355272 See related Ensembl:ENSMUSG00000025162 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 AA409348; 1200006A05Rik; D930010H05Rik Summary This gene encodes a member of the casein kinase I (CKI) family of serine/threonine protein . A highly similar human Expression protein regulates an array of cellular processes by influencing the Wnt and hedgehog signaling pathways. The encoded protein may also be involved in the regulation of apoptosis, circadian rhythm, microtubule dynamics, chromosome segregation, and p53-mediated effects on growth. Alternatively spliced transcript variants encoding different isoforms have been described. [provided by RefSeq, Jul 2008] Orthologs Ubiquitous expression in CNS E14 (RPKM 50.8), whole brain E14.5 (RPKM 50.5) and 28 other tissues See more human all

Genomic context

Location: 11; 11 E2 See Csnk1d in Genome Data Viewer

Exon count: 13

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (120957709..120991350, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (120823055..120852647, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 9 transcripts

Gene: Csnk1d ENSMUSG00000025162

Description casein kinase 1, delta [Source:MGI Symbol;Acc:MGI:1355272] Gene Synonyms 1200006A05Rik Location Chromosome 11: 120,958,990-120,991,330 reverse strand. GRCm38:CM001004.2 About this gene This gene has 9 transcripts (splice variants), 245 orthologues, 11 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Csnk1d- ENSMUST00000018274.9 3675 415aa ENSMUSP00000018274.3 Protein coding CCDS25762 Q9DC28 TSL:1 201 GENCODE basic APPRIS P4

Csnk1d- ENSMUST00000070575.13 3500 409aa ENSMUSP00000070721.7 Protein coding CCDS25763 Q3UBT6 TSL:1 202 Q9DC28 GENCODE basic APPRIS ALT1

Csnk1d- ENSMUST00000239115.1 1823 428aa ENSMUSP00000158953.1 Protein coding - Q3USK2 GENCODE basic 209 APPRIS ALT1

Csnk1d- ENSMUST00000154483.7 655 218aa ENSMUSP00000117472.1 Protein coding - F7A4U8 CDS 5' and 3' 207 incomplete TSL:3

Csnk1d- ENSMUST00000146837.1 689 68aa ENSMUSP00000115055.1 Nonsense mediated - F6VU53 CDS 5' 206 decay incomplete TSL:3

Csnk1d- ENSMUST00000155070.1 604 No - Retained intron - - TSL:1 208 protein

Csnk1d- ENSMUST00000141071.1 1069 No - lncRNA - - TSL:5 205 protein

Csnk1d- ENSMUST00000131375.7 425 No - lncRNA - - TSL:3 203 protein

Csnk1d- ENSMUST00000139267.1 192 No - lncRNA - - TSL:5 204 protein

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52.34 kb Forward strand 120.95Mb 120.96Mb 120.97Mb 120.98Mb 120.99Mb 121.00Mb Slc16a3-201 >protein coding Gm28192-201 >lncRNA (Comprehensive set...

Slc16a3-207 >protein coding

Slc16a3-202 >protein coding

Slc16a3-203 >protein coding

Slc16a3-208 >protein coding

Slc16a3-204 >protein coding

Slc16a3-206 >lncRNA

Slc16a3-205 >retained intron

Contigs AL662901.18 > AL662887.13 > Genes (Comprehensive set... < Csnk1d-209protein coding

< Csnk1d-201protein coding

< Csnk1d-202protein coding

< Csnk1d-203lncRNA < Csnk1d-207protein coding

< Csnk1d-205lncRNA < Csnk1d-206nonsense mediated decay

< Csnk1d-204lncRNA < Csnk1d-208retained intron

Regulatory Build

120.95Mb 120.96Mb 120.97Mb 120.98Mb 120.99Mb 121.00Mb Reverse strand 52.34 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000018274

< Csnk1d-201protein coding

Reverse strand 29.58 kb

ENSMUSP00000018... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Superfamily Protein kinase-like domain superfamily

SMART Protein kinase domain

Pfam Protein kinase domain

PROSITE profiles Protein kinase domain PROSITE patterns Protein kinase, ATP binding site Serine/threonine-protein kinase, active site

PANTHER PTHR11909:SF336

PTHR11909 Gene3D 1.10.510.10

CDD cd14125

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

Variant Legend

synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 415

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

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