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Mouse Apba2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Apba2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Apba2 gene (NCBI Reference Sequence: NM_007461 ; Ensembl: ENSMUSG00000030519 ) is located on Mouse chromosome 7. 15 exons are identified, with the ATG start codon in exon 4 and the TAG stop codon in exon 15 (Transcript: ENSMUST00000032732). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Apba2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-23D18 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: Mice homozygous for disruptions in this gene show a selective deficit in motivated approach behavior, but not in motivated avoidance behavior.

Exon 5 starts from about 42.44% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 18413 bp, and the size of intron 6 for 3'-loxP site insertion: 17965 bp. The size of effective cKO region: ~1617 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 5 6 15 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Apba2 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(8117bp) | A(28.31% 2298) | C(20.28% 1646) | T(27.78% 2255) | G(23.63% 1918)

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% chr7 + 64711182 64714181 3000 browser details YourSeq 347 1671 2055 3000 95.8% chr3 + 88267742 88268124 383 browser details YourSeq 345 1651 2055 3000 94.8% chr18 - 33221027 33221431 405 browser details YourSeq 345 1667 2060 3000 94.7% chr12 + 72081275 72081665 391 browser details YourSeq 344 1671 2060 3000 95.8% chr6 - 53464214 53464603 390 browser details YourSeq 342 1667 2055 3000 94.7% chr6 - 53465700 53466086 387 browser details YourSeq 340 1664 2055 3000 94.6% chr12 - 21020922 21021313 392 browser details YourSeq 339 1671 2055 3000 94.7% chr1 - 88614003 88614384 382 browser details YourSeq 339 1671 2055 3000 94.7% chr6 + 69100145 69100527 383 browser details YourSeq 338 1671 2055 3000 94.6% chrX - 10947806 10948187 382 browser details YourSeq 338 1672 2055 3000 94.6% chr1 - 88615481 88615861 381 browser details YourSeq 338 1671 2055 3000 94.2% chr14 + 87086502 87086883 382 browser details YourSeq 337 1672 2055 3000 93.6% chr7 + 97363530 97363910 381 browser details YourSeq 337 1672 2055 3000 94.0% chr7 + 66313640 66314022 383 browser details YourSeq 337 1656 2055 3000 95.0% chr17 + 64150789 64151398 610 browser details YourSeq 336 1672 2055 3000 94.3% chrX - 57494791 57495170 380 browser details YourSeq 336 1672 2055 3000 94.1% chr3 - 27356266 27356645 380 browser details YourSeq 336 1671 2055 3000 94.2% chr2 - 174910362 174910745 384 browser details YourSeq 336 1672 2055 3000 93.3% chr8 + 57752314 57752691 378 browser details YourSeq 335 1671 2055 3000 94.1% chr9 - 68322680 68323061 382

Note: The 3000 bp section upstream of Exon 5 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% chr7 + 64715799 64718798 3000 browser details YourSeq 161 2346 2717 3000 90.5% chr15 + 81057336 81057710 375 browser details YourSeq 143 2329 2742 3000 85.8% chr10 + 84864916 84865332 417 browser details YourSeq 142 2335 2710 3000 78.9% chr10 + 126820133 126820489 357 browser details YourSeq 140 2352 2711 3000 86.0% chr19 + 57726246 57726603 358 browser details YourSeq 133 2335 2617 3000 85.2% chr7 - 121671294 121671583 290 browser details YourSeq 130 2328 2671 3000 90.2% chr6 + 92317990 92335918 17929 browser details YourSeq 129 2354 2741 3000 89.6% chr11 - 68752315 68752706 392 browser details YourSeq 129 2358 2716 3000 88.2% chr5 + 136777204 136777591 388 browser details YourSeq 127 2349 2667 3000 89.7% chr5 - 116142717 116143039 323 browser details YourSeq 127 2352 2716 3000 89.2% chr11 - 81071223 81071615 393 browser details YourSeq 124 2343 2711 3000 89.9% chrX + 8080230 8080601 372 browser details YourSeq 124 2352 2701 3000 88.0% chr15 + 59298943 59299322 380 browser details YourSeq 123 2444 2867 3000 90.2% chr18 - 53124683 53125168 486 browser details YourSeq 122 2436 2739 3000 87.2% chr15 - 84261436 84261758 323 browser details YourSeq 119 2334 2686 3000 84.7% chr2 + 151851216 151851554 339 browser details YourSeq 116 2447 2706 3000 89.6% chr16 - 92127297 92127599 303 browser details YourSeq 114 2432 2692 3000 89.0% chr1 - 191271725 191272001 277 browser details YourSeq 113 2356 2716 3000 90.1% chr14 - 57174761 57175125 365 browser details YourSeq 113 2450 2741 3000 81.9% chr11 - 109530563 109530837 275

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Apba2 amyloid beta (A4) precursor protein-binding, family A, member 2 [ Mus musculus (house mouse) ] Gene ID: 11784, updated on 14-Aug-2019

Gene summary

Official Symbol Apba2 provided by MGI Official Full Name amyloid beta (A4) precursor protein-binding, family A, member 2 provided by MGI Primary source MGI:MGI:1261791 See related Ensembl:ENSMUSG00000030519 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 X11L; XllL; mXllL; X11-like Expression Biased expression in CNS E18 (RPKM 37.9), cortex adult (RPKM 35.0) and 7 other tissues See more Orthologs human all

Genomic context

Location: 7 C; 7 34.65 cM See Apba2 in Genome Data Viewer

Exon count: 19

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (64501560..64753878)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (71646592..71898764)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 7 transcripts

Gene: Apba2 ENSMUSG00000030519

Description amyloid beta (A4) precursor protein-binding, family A, member 2 [Source:MGI Symbol;Acc:MGI:1261791] Gene Synonyms Mint 2, X11-like, X11L Location Chromosome 7: 64,501,706-64,753,878 forward strand. GRCm38:CM001000.2 About this gene This gene has 7 transcripts (splice variants), 248 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Apba2-201 ENSMUST00000032732.14 3321 750aa ENSMUSP00000032732.8 Protein coding CCDS21335 P98084 TSL:1 GENCODE basic APPRIS P2

Apba2-206 ENSMUST00000206246.1 3027 738aa ENSMUSP00000146038.1 Protein coding - A0A0U1RPM0 TSL:5 GENCODE basic APPRIS ALT2

Apba2-204 ENSMUST00000205613.1 714 81aa ENSMUSP00000146269.1 Protein coding - A0A0U1RQ68 CDS 3' incomplete TSL:3

Apba2-203 ENSMUST00000205604.1 643 102aa ENSMUSP00000146279.1 Protein coding - A0A0U1RQ78 CDS 3' incomplete TSL:2

Apba2-207 ENSMUST00000206630.1 501 79aa ENSMUSP00000146108.1 Protein coding - A0A0U1RPS9 CDS 5' incomplete TSL:3

Apba2-205 ENSMUST00000206061.1 898 No protein - lncRNA - - TSL:1

Apba2-202 ENSMUST00000205551.1 772 No protein - lncRNA - - TSL:5

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272.17 kb Forward strand 64.5Mb 64.6Mb 64.7Mb Genes (Comprehensive set... Apba2-201 >protein coding

Apba2-204 >protein coding Apba2-207 >protein coding

Apba2-203 >protein coding Apba2-202 >lncRNA

Gm45082-201 >TEC Apba2-206 >protein coding

Gm44721-201 >TEC Apba2-205 >lncRNA

Contigs AC140297.3 > AC127306.3 > Genes < Fam189a1-201protein coding (Comprehensive set...

< Fam189a1-202lncRNA

Regulatory Build

64.5Mb 64.6Mb 64.7Mb Reverse strand 272.17 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000032732

252.17 kb Forward strand

Apba2-201 >protein coding

ENSMUSP00000032... MobiDB lite Low complexity (Seg) Superfamily SSF50729 PDZ superfamily

SMART PTB/PI domain PDZ domain

Pfam PTB/PI domain PDZ domain

PROSITE profiles PTB/PI domain PDZ domain

PANTHER PTHR12345

Amyloid beta A4 precursor protein-binding family A member 2 Gene3D PH-like domain superfamily 2.30.42.10 CDD cd01208 cd00992

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

Variant Legend

missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 750

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

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