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Mouse Appbp2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Appbp2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Appbp2 gene (NCBI Reference Sequence: NM_025825 ; Ensembl: ENSMUSG00000018481 ) is located on Mouse chromosome 11. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000018625). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Appbp2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-151A16 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 7.92% of the coding region. The knockout of Exon 2~4 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 18147 bp, and the size of intron 4 for 3'-loxP site insertion: 4127 bp. The size of effective cKO region: ~2789 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 2 3 4 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Appbp2 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(9289bp) | A(28.36% 2634) | C(20.17% 1874) | T(30.78% 2859) | G(20.69% 1922)

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 - 85216775 85219774 3000 browser details YourSeq 1702 1209 2992 3000 98.0% chr4 + 123735357 123743574 8218 browser details YourSeq 1622 1197 3000 3000 96.6% chr17 - 36179321 36181179 1859 browser details YourSeq 1600 1194 3000 3000 95.9% chr17 - 36158198 36160068 1871 browser details YourSeq 1191 1194 2472 3000 96.4% chr6 - 92088687 92089961 1275 browser details YourSeq 749 1223 2501 3000 85.9% chr14 - 61376714 61377926 1213 browser details YourSeq 155 1383 1597 3000 85.8% chr4 - 48009062 48009275 214 browser details YourSeq 86 268 427 3000 88.3% chr12 - 108182333 108182519 187 browser details YourSeq 84 268 428 3000 90.8% chr16 + 31400103 31400466 364 browser details YourSeq 76 338 554 3000 89.7% chr14 - 7746178 7746731 554 browser details YourSeq 76 336 428 3000 92.5% chr13 + 38120232 38120333 102 browser details YourSeq 74 292 431 3000 94.1% chr11 + 29676888 29677034 147 browser details YourSeq 73 331 429 3000 92.1% chr15 + 78766238 78766345 108 browser details YourSeq 72 331 430 3000 89.3% chr11 - 50263797 50263904 108 browser details YourSeq 70 347 542 3000 90.6% chr6 + 40647531 40647822 292 browser details YourSeq 69 296 427 3000 87.3% chr19 + 5552126 5552258 133 browser details YourSeq 69 282 426 3000 83.6% chr17 + 79981855 79981991 137 browser details YourSeq 68 333 429 3000 88.8% chr11 - 118930103 118930208 106 browser details YourSeq 67 333 432 3000 90.6% chr13 - 103939084 103939193 110 browser details YourSeq 67 333 429 3000 89.6% chr11 - 54980880 54981427 548

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% chr11 - 85210986 85213985 3000 browser details YourSeq 137 2361 2550 3000 94.3% chr11 + 3604500 3697103 92604 browser details YourSeq 126 2359 2982 3000 92.7% chr10 + 61754335 62162074 407740 browser details YourSeq 121 2374 2550 3000 93.7% chr1 - 180728888 180881884 152997 browser details YourSeq 117 2356 2538 3000 92.1% chr13 - 14167867 14586564 418698 browser details YourSeq 111 2376 2549 3000 94.5% chr11 - 87830831 88043619 212789 browser details YourSeq 106 2433 2887 3000 79.9% chr4 + 118151657 118151833 177 browser details YourSeq 90 2436 2550 3000 94.3% chr9 + 25467576 25467692 117 browser details YourSeq 86 2435 2547 3000 93.1% chr8 + 25554173 25554288 116 browser details YourSeq 81 2434 2550 3000 87.0% chr7 + 46623101 46623208 108 browser details YourSeq 78 2434 2548 3000 94.5% chr3 + 135668648 135668762 115 browser details YourSeq 78 2427 2547 3000 87.8% chr10 + 92848349 92848464 116 browser details YourSeq 77 2434 2547 3000 94.5% chr12 + 52798539 52798671 133 browser details YourSeq 76 2429 2538 3000 87.3% chr18 + 42024409 42024510 102 browser details YourSeq 75 2427 2547 3000 84.5% chr17 + 17429586 17429698 113 browser details YourSeq 75 2427 2546 3000 86.7% chr11 + 96079599 96079713 115 browser details YourSeq 74 2361 2888 3000 78.9% chrX - 103337603 103338084 482 browser details YourSeq 74 2433 2547 3000 94.2% chr13 + 53316061 53316181 121 browser details YourSeq 72 2434 2542 3000 90.6% chr2 + 154600307 154600413 107 browser details YourSeq 71 2323 2427 3000 83.4% chr2 + 156883136 156883226 91

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: Appbp2 amyloid beta precursor protein (cytoplasmic tail) binding protein 2 [ Mus musculus (house mouse) ] Gene ID: 66884, updated on 10-Oct-2019

Gene summary

Official Symbol Appbp2 provided by MGI Official Full Name amyloid beta precursor protein (cytoplasmic tail) binding protein 2 provided by MGI Primary source MGI:MGI:1914134 See related Ensembl:ENSMUSG00000018481 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as PAT1; AI465480; 1300003O07Rik Expression Ubiquitous expression in ovary adult (RPKM 24.7), placenta adult (RPKM 21.7) and 28 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 C See Appbp2 in Genome Data Viewer

Exon count: 13

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (85191308..85235120, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (85004810..85048622, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 1 transcript

Gene: Appbp2 ENSMUSG00000018481

Description amyloid beta precursor protein (cytoplasmic tail) binding protein 2 [Source:MGI Symbol;Acc:MGI:1914134] Gene Synonyms 1300003O07Rik, PAT1 Location Chromosome 11: 85,187,262-85,235,130 reverse strand. GRCm38:CM001004.2 About this gene This gene has 1 transcript (splice variant), 175 orthologues, 6 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Appbp2-201 ENSMUST00000018625.9 6463 585aa ENSMUSP00000018625.9 Protein coding CCDS25193 Q9DAX9 TSL:1 GENCODE basic APPRIS P1

67.87 kb Forward strand 85.18Mb 85.20Mb 85.22Mb 85.24Mb Genes 1700125H20Rik-202 >protein coding Akirin1-ps-201 >processed pseudogene Appbp2os-202 >pseudogene (Comprehensive set...

1700125H20Rik-201 >protein coding Appbp2os-201 >lncRNA

Contigs AL596183.11 > Genes < Appbp2-201protein coding (Comprehensive set...

Regulatory Build

85.18Mb 85.20Mb 85.22Mb 85.24Mb Reverse strand 67.87 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

pseudogene RNA gene

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Transcript: ENSMUST00000018625

< Appbp2-201protein coding

Reverse strand 47.87 kb

ENSMUSP00000018... Superfamily Tetratricopeptide-like helical domain superfamily SMART Tetratricopeptide repeat Pfam PF13424 PF13374

PROSITE profiles Tetratricopeptide repeat

Tetratricopeptide repeat-containing domain PANTHER Amyloid protein-binding protein 2 Gene3D Tetratricopeptide-like helical domain superfamily

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

Variant Legend synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 585

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

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