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Mouse Asb2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Asb2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Asb2 gene (NCBI Reference Sequence: NM_023049 ; Ensembl: ENSMUSG00000021200 ) is located on Mouse chromosome 12. 10 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000021617). 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 Asb2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-78J12 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 conditional cells activated in the immune system exhibit impaired immature dendritic cell migration.

Exon 4 starts from about 16.4% 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: 2100 bp, and the size of intron 4 for 3'-loxP site insertion: 805 bp. The size of effective cKO region: ~984 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 3 4 5 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Asb2 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(7167bp) | A(24.36% 1746) | C(25.65% 1838) | T(23.33% 1672) | G(26.66% 1911)

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% chr12 - 103336227 103339226 3000 browser details YourSeq 61 39 144 3000 91.6% chr2 - 10115892 10115997 106 browser details YourSeq 41 43 146 3000 70.2% chr16 + 97426337 97426441 105 browser details YourSeq 38 99 146 3000 89.6% chr17 + 51707945 51707992 48 browser details YourSeq 37 52 150 3000 92.9% chr15 + 97419743 97419842 100 browser details YourSeq 35 54 164 3000 82.1% chr12 - 79417886 79417991 106 browser details YourSeq 35 43 85 3000 92.5% chr3 + 28464623 28464667 45 browser details YourSeq 35 1 112 3000 63.9% chr16 + 6957898 6958007 110 browser details YourSeq 34 52 150 3000 94.8% chr1 - 67789079 67789178 100 browser details YourSeq 34 41 82 3000 92.4% chr1 + 189334091 189334141 51 browser details YourSeq 33 2167 2214 3000 86.2% chr13 + 102824395 102824439 45 browser details YourSeq 33 42 92 3000 92.4% chr1 + 30791110 30791162 53 browser details YourSeq 31 43 82 3000 91.7% chr1 + 127567927 127567966 40 browser details YourSeq 30 52 141 3000 96.9% chr2 - 163992585 163992676 92 browser details YourSeq 28 2181 2208 3000 100.0% chr6 - 99269351 99269378 28 browser details YourSeq 28 2186 2213 3000 100.0% chr1 - 136158415 136158442 28 browser details YourSeq 27 2186 2214 3000 89.3% chr13 + 53119537 53119564 28 browser details YourSeq 27 122 152 3000 93.6% chr1 + 188933957 188933987 31 browser details YourSeq 26 52 81 3000 86.3% chr1 - 120288926 120288954 29 browser details YourSeq 25 2180 2206 3000 88.5% chr2 - 57275881 57275906 26

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% chr12 - 103332560 103335559 3000 browser details YourSeq 48 2749 2812 3000 96.3% chr14 - 120620381 120620469 89 browser details YourSeq 34 2779 2813 3000 100.0% chr8 + 102506489 102506524 36 browser details YourSeq 33 2779 2816 3000 94.8% chr16 + 60274908 60274946 39 browser details YourSeq 32 2776 2817 3000 88.1% chrX + 122875471 122875512 42 browser details YourSeq 31 2727 2768 3000 97.0% chr2 + 28712743 28712837 95 browser details YourSeq 29 2778 2815 3000 78.8% chr7 + 72355948 72355981 34 browser details YourSeq 27 2752 2779 3000 100.0% chr12 + 28056225 28056254 30 browser details YourSeq 26 2259 2285 3000 100.0% chr6 - 108538961 108538988 28 browser details YourSeq 25 2778 2805 3000 96.3% chr19 - 24567532 24567563 32 browser details YourSeq 25 2744 2768 3000 100.0% chr18 + 6884346 6884370 25 browser details YourSeq 24 2715 2738 3000 100.0% chr2 + 70878333 70878356 24 browser details YourSeq 22 2749 2770 3000 100.0% chr6 + 8622768 8622789 22 browser details YourSeq 22 2796 2817 3000 100.0% chr5 + 121199285 121199306 22 browser details YourSeq 22 457 478 3000 100.0% chr11 + 114635226 114635247 22 browser details YourSeq 21 2748 2768 3000 100.0% chr4 - 24520424 24520444 21 browser details YourSeq 21 2748 2768 3000 100.0% chr12 - 4944009 4944029 21 browser details YourSeq 20 2749 2768 3000 100.0% chr15 - 98308880 98308899 20 browser details YourSeq 20 2748 2767 3000 100.0% chr12 - 50939440 50939459 20 browser details YourSeq 20 2749 2768 3000 100.0% chr12 + 15579239 15579258 20

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: Asb2 ankyrin repeat and SOCS box-containing 2 [ Mus musculus (house mouse) ] Gene ID: 65256, updated on 24-Oct-2019

Gene summary

Official Symbol Asb2 provided by MGI Official Full Name ankyrin repeat and SOCS box-containing 2 provided by MGI Primary source MGI:MGI:1929743 See related Ensembl:ENSMUSG00000021200 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 1110008E15Rik Expression Biased expression in heart adult (RPKM 165.8), spleen adult (RPKM 62.0) and 11 other tissues See more Orthologs human all

Genomic context

Location: 12 E; 12 52.9 cM See Asb2 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (103321142..103356001, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (104559352..104594211, complement)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 4 transcripts

Gene: Asb2 ENSMUSG00000021200

Description ankyrin repeat and SOCS box-containing 2 [Source:MGI Symbol;Acc:MGI:1929743] Gene Synonyms 1110008E15Rik Location Chromosome 12: 103,321,142-103,356,001 reverse strand. GRCm38:CM001005.2 About this gene This gene has 4 transcripts (splice variants), 200 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Asb2-201 ENSMUST00000021617.13 2690 634aa ENSMUSP00000021617.7 Protein coding CCDS26128 Q8K0L0 TSL:1 GENCODE basic APPRIS P2

Asb2-204 ENSMUST00000149431.1 2283 586aa ENSMUSP00000117595.1 Protein coding - Q8K0L0 TSL:1 GENCODE basic APPRIS ALT2

Asb2-202 ENSMUST00000127447.1 2399 No protein - Retained intron - - TSL:1

Asb2-203 ENSMUST00000135694.1 383 No protein - lncRNA - - TSL:3

54.86 kb Forward strand 103.32Mb 103.33Mb 103.34Mb 103.35Mb 103.36Mb Genes Fam181a-203 >protein coding Gm15523-201 >lncRNA (Comprehensive set...

Fam181a-202 >protein coding

Fam181a-201 >protein coding

Contigs AC160131.2 >

Genes (Comprehensive set... < Gm29508-201lncRNA < Asb2-201protein coding

< Gm29508-202lncRNA < Asb2-204protein coding

< Asb2-202retained intron

< Asb2-203lncRNA

Regulatory Build

103.32Mb 103.33Mb 103.34Mb 103.35Mb 103.36Mb Reverse strand 54.86 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000021617

< Asb2-201protein coding

Reverse strand 34.86 kb

ENSMUSP00000021... MobiDB lite Low complexity (Seg) Superfamily Ankyrin repeat-containing domain superfamily SOCS box-like domain superfamily

SMART Ankyrin repeat SOCS box domain

SOCS box domain Prints Ankyrin repeat Pfam PF13637 Ankyrin repeat-containing domain SOCS box domain

Ankyrin repeat PROSITE profiles Ankyrin repeat-containing domain SOCS box domain

Ubiquitin interacting motif Ankyrin repeat PANTHER PTHR24173:SF33

PTHR24173 Gene3D 1.25.40.960 1.10.750.20

Ankyrin repeat-containing domain superfamily

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

Variant Legend

missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 634

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

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