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Mouse Calcoco1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Calcoco1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Calcoco1 gene (NCBI Reference Sequence: NM_026192 ; Ensembl: ENSMUSG00000023055 ) is located on Mouse chromosome 15. 15 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 15 (Transcript: ENSMUST00000023818). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Calcoco1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-449K20 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 4 starts from about 12.54% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 980 bp, and the size of intron 4 for 3'-loxP site insertion: 1414 bp. The size of effective cKO region: ~691 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 2 3 4 5 6 15 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Calcoco1 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7191bp) | A(27.12% 1950) | C(22.63% 1627) | T(25.67% 1846) | G(24.59% 1768)

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% chr15 - 102718244 102721243 3000 browser details YourSeq 254 1898 2429 3000 87.7% chr16 + 20667962 20668355 394 browser details YourSeq 249 1897 2444 3000 90.6% chr5 - 110239999 110240559 561 browser details YourSeq 238 1900 2437 3000 85.9% chr12 - 54084793 54085238 446 browser details YourSeq 238 1898 2436 3000 89.1% chr1 + 181875514 181876491 978 browser details YourSeq 234 1899 2429 3000 91.9% chr13 - 55216818 55217474 657 browser details YourSeq 233 1895 2411 3000 90.7% chr10 + 20292421 20293051 631 browser details YourSeq 225 1900 2444 3000 91.0% chr9 + 58244647 58245204 558 browser details YourSeq 191 1898 2436 3000 81.6% chr1 - 58563302 58563680 379 browser details YourSeq 180 1892 2468 3000 85.2% chr1 - 59542937 59543442 506 browser details YourSeq 175 1898 2361 3000 90.8% chr7 + 126507114 126507781 668 browser details YourSeq 175 1971 2401 3000 94.0% chr1 + 134476275 134476851 577 browser details YourSeq 173 1900 2415 3000 88.5% chr17 - 88499102 88499622 521 browser details YourSeq 163 1898 2392 3000 86.1% chr7 + 143581252 143581688 437 browser details YourSeq 159 1900 2335 3000 90.9% chr10 + 75225833 75226457 625 browser details YourSeq 157 1902 2332 3000 83.1% chr17 - 47510072 47510317 246 browser details YourSeq 156 1900 2392 3000 82.1% chr7 - 28281845 28282129 285 browser details YourSeq 156 1898 2457 3000 83.7% chr1 + 135335870 135336129 260 browser details YourSeq 153 1898 2075 3000 93.1% chr16 - 20346355 20346531 177 browser details YourSeq 152 1899 2308 3000 91.9% chr11 - 80344093 80344681 589

Note: The 3000 bp section upstream of Exon 4 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% chr15 - 102714553 102717552 3000 browser details YourSeq 134 2178 2628 3000 79.1% chrX + 8269231 8269486 256 browser details YourSeq 107 2509 2896 3000 80.8% chr9 - 61587274 61587517 244 browser details YourSeq 107 2469 2629 3000 90.1% chr7 - 26675721 26675880 160 browser details YourSeq 105 2460 2630 3000 83.9% chr9 + 110419223 110419384 162 browser details YourSeq 103 2465 2628 3000 86.6% chr14 - 96506585 96506744 160 browser details YourSeq 102 2459 2627 3000 89.0% chr13 - 80397016 80397183 168 browser details YourSeq 102 2465 2614 3000 82.6% chrX + 12199238 12199386 149 browser details YourSeq 101 2460 2629 3000 91.9% chr11 - 71809330 71809512 183 browser details YourSeq 100 2509 2631 3000 87.5% chr4 + 134794734 134794853 120 browser details YourSeq 98 2461 2584 3000 90.4% chr11 - 34954154 34954276 123 browser details YourSeq 98 2460 2626 3000 80.2% chr5 + 115585957 115586111 155 browser details YourSeq 97 2478 2628 3000 90.8% chr4 + 68759455 68759662 208 browser details YourSeq 94 2460 2631 3000 93.7% chr7 - 21434156 21434378 223 browser details YourSeq 94 2509 2633 3000 91.4% chr17 - 35117164 35117291 128 browser details YourSeq 93 2460 2580 3000 91.7% chr2 - 180325132 180325251 120 browser details YourSeq 93 2509 2619 3000 91.9% chr8 + 40932786 40932896 111 browser details YourSeq 92 2506 2627 3000 88.3% chr5 - 107065809 107065928 120 browser details YourSeq 92 2509 2628 3000 89.1% chrX + 140112753 140112870 118 browser details YourSeq 92 2474 2620 3000 85.0% chrX + 11478543 11478689 147

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: Calcoco1 calcium binding and coiled coil domain 1 [ Mus musculus (house mouse) ] Gene ID: 67488, updated on 12-Aug-2019

Gene summary

Official Symbol Calcoco1 provided by MGI Official Full Name calcium binding and coiled coil domain 1 provided by MGI Primary source MGI:MGI:1914738 See related Ensembl:ENSMUSG00000023055 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 Cocoa; Gcap11; mKIAA1536; 1810009B06Rik Expression Ubiquitous expression in lung adult (RPKM 33.2), heart adult (RPKM 30.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 15 F3; 15 See Calcoco1 in Genome Data Viewer

Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (102706777..102722178, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (102537208..102552609, complement)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 9 transcripts

Gene: Calcoco1 ENSMUSG00000023055

Description calcium binding and coiled coil domain 1 [Source:MGI Symbol;Acc:MGI:1914738] Gene Synonyms 1810009B06Rik, CoCoA, Gcap11 Location Chromosome 15: 102,706,777-102,722,178 reverse strand. GRCm38:CM001008.2 About this gene This gene has 9 transcripts (splice variants), 231 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Calcoco1- ENSMUST00000023818.10 2824 691aa ENSMUSP00000023818.3 Protein coding CCDS27889 Q8CGU1 TSL:1 201 GENCODE basic APPRIS P1

Calcoco1- ENSMUST00000171838.1 2968 633aa ENSMUSP00000130170.1 Protein coding - E9Q7U2 TSL:1 204 GENCODE basic

Calcoco1- ENSMUST00000229231.1 2226 606aa ENSMUSP00000155767.1 Protein coding - A0A2R8VKP5 GENCODE 205 basic

Calcoco1- ENSMUST00000230035.1 1620 533aa ENSMUSP00000155446.1 Protein coding - A0A2R8VHU8 CDS 3' 207 incomplete

Calcoco1- ENSMUST00000168112.7 1095 232aa ENSMUSP00000128307.1 Protein coding - F6Q5V4 CDS 5' 203 incomplete TSL:5

Calcoco1- ENSMUST00000229398.1 921 68aa ENSMUSP00000155415.1 Nonsense mediated - A0A2R8VKE2 - 206 decay

Calcoco1- ENSMUST00000230374.1 1732 No - Retained intron - - - 208 protein

Calcoco1- ENSMUST00000166943.1 863 No - Retained intron - - TSL:2 202 protein

Calcoco1- ENSMUST00000230702.1 462 No - Retained intron - - - 209 protein

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35.40 kb Forward strand

102.70Mb 102.71Mb 102.72Mb 102.73Mb Contigs < AC123870.3 < AC124532.4 Genes (Comprehensive set... < Calcoco1-201protein coding

< Calcoco1-203protein coding< Calcoco1-208retained intron

< Calcoco1-205protein coding

< Calcoco1-204protein coding

< Calcoco1-209retained intron

< Calcoco1-207protein coding

< Calcoco1-202retained intron

< Calcoco1-206nonsense mediated decay

Regulatory Build

102.70Mb 102.71Mb 102.72Mb 102.73Mb Reverse strand 35.40 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000023818

< Calcoco1-201protein coding

Reverse strand 15.40 kb

ENSMUSP00000023... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57997 Pfam SKICH domain Calcium binding and coiled-coil domain-like CALCOCO1/2, zinc finger-C2H2 domain

PANTHER PTHR31915:SF5

PTHR31915 Gene3D 2.60.40.2840 3.30.160.550

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 691

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

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