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Mouse Baiap3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Baiap3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Baiap3 (NCBI Reference Sequence: NM_001163270 ; Ensembl: ENSMUSG00000047507 ) is located on Mouse 17. 34 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 34 (Transcript: ENSMUST00000182056). Exon 6~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Baiap3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-333E5 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 a null allele are viable and fertile but exhibit increased PTZ-induced seizure propensity, as well as increased novelty-induced anxiety in both genders, with a more pronounced effect in females, and a faster developmentof tolerance to benzodiazepines in male mice.

Exon 6 starts from about 11.68% of the coding region. The knockout of Exon 6~8 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 445 bp, and the size of intron 8 for 3'-loxP site insertion: 523 bp. The size of effective cKO region: ~1045 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 5 6 7 8 9 10 11 12 13 14 15 34 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Baiap3 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(7517bp) | A(22.19% 1668) | C(25.17% 1892) | T(22.44% 1687) | G(30.2% 2270)

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% chr17 - 25250878 25253877 3000 browser details YourSeq 23 2871 2896 3000 96.0% chr7 + 139592481 139592507 27 browser details YourSeq 22 549 570 3000 100.0% chr1 + 82635665 82635686 22 browser details YourSeq 21 59 79 3000 100.0% chr2 - 77206981 77207001 21 browser details YourSeq 21 2818 2838 3000 100.0% chr16 - 89702620 89702640 21 browser details YourSeq 21 1507 1528 3000 100.0% chr10 - 26654549 26654571 23 browser details YourSeq 21 1338 1358 3000 100.0% chr7 + 83514271 83514291 21 browser details YourSeq 21 2145 2165 3000 100.0% chr13 + 102771421 102771441 21

Note: The 3000 bp section upstream of Exon 6 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% chr17 - 25246833 25249832 3000 browser details YourSeq 26 505 537 3000 82.2% chr1 + 67789414 67789443 30 browser details YourSeq 23 1 24 3000 100.0% chr5 + 129593486 129593511 26 browser details YourSeq 22 261 282 3000 100.0% chrX - 110461110 110461131 22 browser details YourSeq 22 1763 1784 3000 100.0% chr11 - 69309391 69309412 22 browser details YourSeq 22 976 1000 3000 95.9% chr4 + 126415731 126415756 26 browser details YourSeq 22 972 993 3000 100.0% chr4 + 94517290 94517311 22 browser details YourSeq 21 2218 2240 3000 95.7% chr1 - 47873614 47873636 23 browser details YourSeq 21 2109 2129 3000 100.0% chr15 + 83342171 83342191 21 browser details YourSeq 21 262 282 3000 100.0% chr11 + 92164271 92164291 21 browser details YourSeq 20 1770 1789 3000 100.0% chr1 + 61025543 61025562 20

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Baiap3 BAI1-associated protein 3 [ Mus musculus (house mouse) ] Gene ID: 545192, updated on 10-Oct-2019

Gene summary

Official Symbol Baiap3 provided by MGI Official Full Name BAI1-associated protein 3 provided by MGI Primary source MGI:MGI:2685783 See related Ensembl:ENSMUSG00000047507 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 Bap3; Gm937 Expression Broad expression in thymus adult (RPKM 8.6), CNS E18 (RPKM 8.4) and 24 other tissues See more Orthologs human all

Genomic context

Location: 17; 17 A3.3 See Baiap3 in Genome Data Viewer

Exon count: 36

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (25242659..25256364, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (25379604..25393309, complement)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 9 transcripts

Gene: Baiap3 ENSMUSG00000047507

Description BAI1-associated protein 3 [Source:MGI Symbol;Acc:MGI:2685783] Gene Synonyms LOC381076 Location Chromosome 17: 25,242,659-25,256,364 reverse strand. GRCm38:CM001010.2 About this gene This gene has 9 transcripts (splice variants), 153 orthologues, 1 paralogue, 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

Baiap3-202 ENSMUST00000182056.7 4645 1150aa ENSMUSP00000138188.1 Protein coding CCDS50033 S4R1E7 TSL:1 GENCODE basic APPRIS P2

Baiap3-206 ENSMUST00000182825.7 3862 1114aa ENSMUSP00000138254.1 Protein coding - S4R1K2 TSL:5 GENCODE basic

Baiap3-204 ENSMUST00000182435.7 3758 1122aa ENSMUSP00000138796.1 Protein coding - S4R2U8 TSL:5 GENCODE basic APPRIS ALT2

Baiap3-201 ENSMUST00000169109.2 3630 1127aa ENSMUSP00000129854.2 Protein coding - E9Q350 TSL:5 GENCODE basic APPRIS ALT2

Baiap3-205 ENSMUST00000182696.7 499 90aa ENSMUSP00000138454.1 Protein coding - S4R212 CDS 5' incomplete TSL:5

Baiap3-203 ENSMUST00000182126.1 1295 No protein - Retained intron - - TSL:1

Baiap3-207 ENSMUST00000182903.1 1020 No protein - Retained intron - - TSL:5

Baiap3-209 ENSMUST00000182978.1 608 No protein - Retained intron - - TSL:3

Baiap3-208 ENSMUST00000182922.1 505 No protein - Retained intron - - TSL:3

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33.71 kb Forward strand 25.24Mb 25.25Mb 25.26Mb Unkl-202 >protein coding Tsr3-201 >protein coding (Comprehensive set...

Unkl-204 >protein coding

Unkl-201 >protein coding

Contigs AC130711.3 > AC122454.4 > Genes (Comprehensive set... < Gnptg-202protein coding < Baiap3-202protein coding < Ube2i-204retained intron

< Gnptg-201protein coding < Baiap3-203retained intron < Baiap3-209retained intron < Ube2i-201protein coding

< Baiap3-206protein coding < Ube2i-208protein coding

< Baiap3-205protein coding < Ube2i-211protein coding

< Baiap3-204protein coding < Ube2i-206protein coding

< Baiap3-201protein coding < Ube2i-214protein coding

< Baiap3-208retained intron < Ube2i-213protein coding

< Mir3547-201miRNA < Ube2i-212protein coding

< Baiap3-207retained intron < Ube2i-210protein coding

Regulatory Build

25.24Mb 25.25Mb 25.26Mb Reverse strand 33.71 kb

Regulation Legend CTCF 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: ENSMUST00000182056

< Baiap3-202protein coding

Reverse strand 13.71 kb

ENSMUSP00000138... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF49562 SMART C2 domain Pfam C2 domain PROSITE profiles Munc13 homology 1 Mammalian uncoordinated homology 13, domain 2

C2 domain PANTHER PTHR45999:SF1

PTHR45999 Gene3D 1.10.357.50 C2 domain superfamily

CDD cd08676 cd04009

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1000 1150

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

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