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Mouse Ckap2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ckap2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ckap2 (NCBI Reference Sequence: NM_001004140 ; Ensembl: ENSMUSG00000037725 ) is located on Mouse 8. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000046916). 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 Ckap2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-299I15 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 5.52% 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: 6088 bp, and the size of intron 2 for 3'-loxP site insertion: 1774 bp. The size of effective cKO region: ~582 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 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ckap2 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(7082bp) | A(28.62% 2027) | C(20.29% 1437) | T(29.98% 2123) | G(21.11% 1495)

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% chr8 - 22179846 22182845 3000 browser details YourSeq 136 1847 1988 3000 96.5% chr8 - 22185215 22185355 141 browser details YourSeq 84 145 264 3000 88.2% chr1 + 73933874 73934018 145 browser details YourSeq 77 150 263 3000 87.2% chr18 - 21087938 21088084 147 browser details YourSeq 72 167 264 3000 87.5% chr12 - 101088618 101088728 111 browser details YourSeq 72 146 241 3000 88.5% chr6 + 116200277 116200403 127 browser details YourSeq 71 149 241 3000 89.3% chr7 + 102112625 102112748 124 browser details YourSeq 71 145 241 3000 87.7% chr4 + 95526385 95526512 128 browser details YourSeq 71 145 241 3000 90.0% chr2 + 11728246 11728373 128 browser details YourSeq 70 149 241 3000 89.9% chr11 - 97386795 97386918 124 browser details YourSeq 70 149 241 3000 89.9% chr11 - 33638498 33638621 124 browser details YourSeq 70 142 241 3000 89.1% chr1 - 134356781 134356912 132 browser details YourSeq 69 158 256 3000 89.1% chr4 + 27832454 27832579 126 browser details YourSeq 68 150 249 3000 88.9% chr10 - 88986367 88986498 132 browser details YourSeq 68 158 241 3000 91.7% chr18 + 44687264 44687378 115 browser details YourSeq 68 167 262 3000 87.8% chr10 + 80009477 80009589 113 browser details YourSeq 67 167 261 3000 86.2% chr1 + 182123432 182123542 111 browser details YourSeq 65 167 264 3000 89.2% chr11 - 5388898 5389009 112 browser details YourSeq 64 169 262 3000 89.1% chr1 - 191757441 191757551 111 browser details YourSeq 64 139 566 3000 90.0% chr5 + 117311905 117312418 514

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% chr8 - 22176264 22179263 3000 browser details YourSeq 676 1523 2490 3000 87.8% chr5 - 49901208 49902113 906 browser details YourSeq 609 1523 2508 3000 90.7% chr2 - 57273446 57275438 1993 browser details YourSeq 57 2810 2912 3000 87.0% chr1 + 84906428 84906528 101 browser details YourSeq 54 2819 2912 3000 82.0% chr4 + 149709628 149709710 83 browser details YourSeq 53 2817 2893 3000 80.0% chr4 - 155742819 155742893 75 browser details YourSeq 53 2807 2904 3000 80.0% chr10 + 125800094 125800187 94 browser details YourSeq 52 2810 2917 3000 72.0% chr12 - 104980724 104980830 107 browser details YourSeq 51 2802 2923 3000 77.8% chr1 - 121132898 121133017 120 browser details YourSeq 49 2819 2907 3000 75.0% chr19 + 5373494 5373581 88 browser details YourSeq 49 2818 2903 3000 84.8% chr10 + 67052457 67052539 83 browser details YourSeq 48 2810 2908 3000 87.5% chr15 - 93278490 93278586 97 browser details YourSeq 48 2819 2899 3000 74.4% chr2 + 118434000 118434077 78 browser details YourSeq 46 825 899 3000 91.1% chr12 + 83896342 83896488 147 browser details YourSeq 46 2819 2904 3000 91.0% chr1 + 58562680 58562766 87 browser details YourSeq 44 965 1223 3000 91.0% chr11 - 73721626 73721890 265 browser details YourSeq 44 2817 2911 3000 73.8% chr1 - 86558985 86559077 93 browser details YourSeq 44 758 837 3000 94.0% chr11 + 90084509 90084593 85 browser details YourSeq 43 2817 2903 3000 79.1% chr7 - 98287223 98287307 85 browser details YourSeq 43 2807 2888 3000 94.0% chr13 - 58116655 58117145 491

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

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Gene and information: Ckap2 associated protein 2 [ Mus musculus (house mouse) ] Gene ID: 80986, updated on 12-Aug-2019

Gene summary

Official Symbol Ckap2 provided by MGI Official Full Name cytoskeleton associated protein 2 provided by MGI Primary source MGI:MGI:1931797 See related Ensembl:ENSMUSG00000037725 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 LB1; AI461656; AW228814 Expression Biased expression in CNS E11.5 (RPKM 15.6), liver E14 (RPKM 9.6) and 13 other tissues See more Orthologs human all

Genomic context

Location: 8; 8 A2 See Ckap2 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (22168149..22185819, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (23278624..23296291, complement)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 3 transcripts

Gene: Ckap2 ENSMUSG00000037725

Description cytoskeleton associated protein 2 [Source:MGI Symbol;Acc:MGI:1931797] Gene Synonyms LB1 Location Chromosome 8: 22,168,160-22,185,819 reverse strand. GRCm38:CM001001.2 About this gene This gene has 3 transcripts (splice variants), 188 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ckap2-201 ENSMUST00000046916.8 2593 664aa ENSMUSP00000039518.7 Protein coding CCDS22170 Q3V1H1 TSL:1 GENCODE basic APPRIS P1

Ckap2-203 ENSMUST00000211629.1 987 No protein - Retained intron - - TSL:2

Ckap2-202 ENSMUST00000211045.1 683 No protein - Retained intron - - TSL:2

37.66 kb Forward strand

22.16Mb 22.17Mb 22.18Mb 22.19Mb Vps36-201 >protein coding (Comprehensive set...

Contigs AC117665.6 >

Genes (Comprehensive set... < Nek3-202protein coding < Ckap2-201protein coding

< Nek3-201protein coding < Ckap2-203retained intron < Ckap2-202retained intron

< Nek3-204protein coding

Regulatory Build

22.16Mb 22.17Mb 22.18Mb 22.19Mb Reverse strand 37.66 kb

Regulation Legend CTCF Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000046916

< Ckap2-201protein coding

Reverse strand 17.66 kb

ENSMUSP00000039... MobiDB lite Low complexity (Seg) Pfam Cytoskeleton-associated protein 2, C-terminal PANTHER Cytoskeleton-associated protein 2 family

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

Variant Legend

inframe insertion inframe deletion missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 664

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

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