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Mouse Cttnbp2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cttnbp2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cttnbp2 gene (NCBI Reference Sequence: NM_080285 ; Ensembl: ENSMUSG00000000416 ) is located on Mouse chromosome 6. 23 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 23 (Transcript: ENSMUST00000090601). 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 Cttnbp2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-86J16 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 8.33% 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: 12801 bp, and the size of intron 4 for 3'-loxP site insertion: 6177 bp. The size of effective cKO region: ~2127 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 23 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cttnbp2 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(8627bp) | A(27.5% 2372) | C(21.79% 1880) | T(29.63% 2556) | G(21.08% 1819)

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% chr6 - 18435697 18438696 3000 browser details YourSeq 216 1506 2112 3000 91.3% chr9 - 104806873 105063154 256282 browser details YourSeq 138 1504 1651 3000 96.7% chr4 - 84176730 84176877 148 browser details YourSeq 137 1452 1647 3000 87.1% chr1 + 39150698 39150859 162 browser details YourSeq 135 1504 1663 3000 96.0% chr12 + 77279384 77279543 160 browser details YourSeq 131 1504 1646 3000 95.9% chr6 + 119360539 119360681 143 browser details YourSeq 130 1504 1651 3000 94.0% chr12 - 56890604 56890751 148 browser details YourSeq 129 1504 1646 3000 93.7% chr7 + 101292545 101292686 142 browser details YourSeq 128 1507 1643 3000 94.9% chr7 + 41689287 41689421 135 browser details YourSeq 128 1504 1643 3000 95.8% chr19 + 54699166 54699305 140 browser details YourSeq 126 1504 1647 3000 93.8% chr7 - 19116784 19116927 144 browser details YourSeq 126 1511 1649 3000 95.7% chr18 + 11683157 11683296 140 browser details YourSeq 126 1504 1643 3000 95.0% chr11 + 29630233 29630372 140 browser details YourSeq 125 1506 1647 3000 94.4% chr10 - 80551141 80551299 159 browser details YourSeq 125 1504 1647 3000 95.0% chr12 + 65756689 65756847 159 browser details YourSeq 125 1507 1647 3000 94.4% chr10 + 77615576 77615716 141 browser details YourSeq 125 1504 1643 3000 95.0% chr1 + 74724510 74724650 141 browser details YourSeq 124 1506 1647 3000 93.7% chr8 - 125090834 125090975 142 browser details YourSeq 124 1504 1639 3000 95.6% chr3 - 110879882 110880017 136 browser details YourSeq 124 1507 1642 3000 95.6% chr15 - 4444510 4444645 136

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% chr6 - 18430570 18433569 3000 browser details YourSeq 111 2856 3000 3000 91.4% chr13 - 49407354 49407507 154 browser details YourSeq 100 2862 3000 3000 84.1% chr15 - 47534484 47534601 118 browser details YourSeq 98 2863 3000 3000 83.1% chr19 - 20853631 20853749 119 browser details YourSeq 95 2862 2987 3000 95.5% chr9 - 90049470 90049607 138 browser details YourSeq 93 2878 2992 3000 95.5% chr11 - 30648191 30648318 128 browser details YourSeq 90 2869 2991 3000 86.0% chr1 - 29023579 29023692 114 browser details YourSeq 88 2888 3000 3000 92.1% chr11 + 47416317 47416435 119 browser details YourSeq 86 2701 3000 3000 82.9% chr6 - 37797617 37797875 259 browser details YourSeq 83 2859 2955 3000 95.8% chr4 - 44393850 44393958 109 browser details YourSeq 79 2869 2974 3000 88.3% chr1 - 29023253 29023353 101 browser details YourSeq 72 2862 2962 3000 79.1% chr6 + 102033839 102033927 89 browser details YourSeq 72 2856 2941 3000 85.0% chr1 + 191951297 191951376 80 browser details YourSeq 69 2864 2977 3000 83.2% chr6 - 37797527 37797630 104 browser details YourSeq 68 2920 3000 3000 92.6% chr5 + 17989765 17989846 82 browser details YourSeq 66 2918 3000 3000 84.9% chr9 - 90049646 90049724 79 browser details YourSeq 62 2881 2966 3000 91.8% chr4 + 22899462 22899569 108 browser details YourSeq 61 2913 3000 3000 76.4% chr17 - 19983329 19983401 73 browser details YourSeq 60 2861 2944 3000 90.5% chr17 - 19983163 19983282 120 browser details YourSeq 60 2931 3000 3000 88.1% chr15 + 52562360 52562426 67

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: Cttnbp2 cortactin binding protein 2 [ Mus musculus (house mouse) ] Gene ID: 30785, updated on 12-Aug-2019

Gene summary

Official Symbol Cttnbp2 provided by MGI Official Full Name cortactin binding protein 2 provided by MGI Primary source MGI:MGI:1353467 See related Ensembl:ENSMUSG00000000416 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 ORF4; Cortbp2; AU040881; mKIAA1758; 6430526E05; 3010022N24Rik; 4732477G22Rik; 9130022E09Rik Expression Biased expression in frontal lobe adult (RPKM 16.8), cortex adult (RPKM 13.4) and 13 other tissues See more Orthologs human all

Genomic context

Location: 6; 6 A2 See Cttnbp2 in Genome Data Viewer

Exon count: 25

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (18366471..18515217, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (18316477..18464825, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 9 transcripts

Gene: Cttnbp2 ENSMUSG00000000416

Description cortactin binding protein 2 [Source:MGI Symbol;Acc:MGI:1353467] Gene Synonyms 3010022N24Rik, 4732477G22Rik, 9130022E09Rik, Cortbp2, ORF4 Location Chromosome 6: 18,366,478-18,514,843 reverse strand. GRCm38:CM000999.2 About this gene This gene has 9 transcripts (splice variants), 213 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cttnbp2- ENSMUST00000090601.11 5183 1648aa ENSMUSP00000088089.5 Protein coding CCDS39432 B9EJA2 TSL:1 201 GENCODE basic APPRIS P1

Cttnbp2- ENSMUST00000146775.7 4224 1139aa ENSMUSP00000119383.1 Protein coding - F6Q1W8 CDS 5' 207 incomplete TSL:1

Cttnbp2- ENSMUST00000141581.1 580 193aa ENSMUSP00000123162.1 Protein coding - F6XSM7 CDS 5' and 3' 205 incomplete TSL:5

Cttnbp2- ENSMUST00000129669.7 575 160aa ENSMUSP00000116878.1 Protein coding - D3Z1E3 CDS 3' 202 incomplete TSL:5

Cttnbp2- ENSMUST00000142963.2 328 94aa ENSMUSP00000122590.1 Protein coding - D3Z551 CDS 3' 206 incomplete TSL:3

Cttnbp2- ENSMUST00000148602.7 5462 630aa ENSMUSP00000118432.1 Nonsense mediated - B9EJA2 TSL:1 208 decay R7RU63

Cttnbp2- ENSMUST00000152499.7 4391 No - Retained intron - - TSL:1 209 protein

Cttnbp2- ENSMUST00000139557.1 3982 No - Retained intron - - TSL:1 203 protein

Cttnbp2- ENSMUST00000140416.1 620 No - Retained intron - - TSL:2 204 protein

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168.37 kb Forward strand 18.40Mb 18.45Mb 18.50Mb Genes Gm15594-201 >lncRNA (Comprehensive set...

Contigs AC158663.2 > AC027654.3 > Genes (Comprehensive set... < Cttnbp2-209retained intron < Gm43843-201TEC < Cttnbp2-204retained intron

< Cttnbp2-207protein coding < Cttnbp2-202protein coding

< Cttnbp2-208nonsense mediated decay

< Cttnbp2-201protein coding

< Cttnbp2-205protein coding < Cttnbp2-203retained intron

< Cttnbp2-206protein coding

< Gm26233-201miRNA

Regulatory Build

18.40Mb 18.45Mb 18.50Mb Reverse strand 168.37 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000090601

< Cttnbp2-201protein coding

Reverse strand 147.60 kb

ENSMUSP00000088... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Ankyrin repeat-containing domain superfamily SMART Ankyrin repeat Pfam Cortactin-binding protein-2, N-terminal Ankyrin repeat-containing domain

Ankyrin repeat PROSITE profiles Ankyrin repeat-containing domain

Ankyrin repeat PANTHER PTHR24166

PTHR24166:SF27 Gene3D Ankyrin repeat-containing domain superfamily

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

Variant Legend inframe insertion missense variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1648

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

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