https://www.alphaknockout.com

Mouse Asb13 Knockout Project (CRISPR/Cas9)

Objective: To create a Asb13 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Asb13 gene (NCBI Reference Sequence: NM_178283 ; Ensembl: ENSMUSG00000033781 ) is located on Mouse chromosome 13. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000042288). Exon 2 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note:

Exon 2 starts from about 5.28% of the coding region. Exon 2 covers 22.54% of the coding region. The size of effective KO region: ~188 bp. The KO 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 2 6

Legends Exon of mouse Asb13 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 2 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 1222 bp section downstream of Exon 2 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(24.65% 493) | C(22.1% 442) | T(32.25% 645) | G(21.0% 420)

Note: The 2000 bp section upstream of Exon 2 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(1222bp) | A(24.39% 298) | C(21.28% 260) | T(29.38% 359) | G(24.96% 305)

Note: The 1222 bp section downstream of Exon 2 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 2000 1 2000 2000 100.0% chr13 + 3640066 3642065 2000 browser details YourSeq 163 1371 1538 2000 98.9% chr6 - 4646717 4646896 180 browser details YourSeq 161 1358 1533 2000 94.8% chr7 + 64201051 64201224 174 browser details YourSeq 158 1371 1533 2000 98.8% chrX - 140757169 140757340 172 browser details YourSeq 158 1367 1532 2000 97.6% chrX - 22915584 22915749 166 browser details YourSeq 158 1371 1538 2000 95.8% chr9 - 62217510 62217674 165 browser details YourSeq 158 1371 1538 2000 98.2% chr4 + 145446143 145446316 174 browser details YourSeq 157 1371 1533 2000 98.8% chr19 - 8730543 8730712 170 browser details YourSeq 157 1368 1529 2000 99.4% chr14 - 13908148 13908332 185 browser details YourSeq 157 1375 1559 2000 93.5% chr18 + 49403143 49403327 185 browser details YourSeq 156 1371 1531 2000 99.4% chr15 + 101274835 101274996 162 browser details YourSeq 155 1373 1533 2000 98.8% chr1 - 153538788 153538953 166 browser details YourSeq 154 1370 1533 2000 97.6% chr4 - 83415227 83415393 167 browser details YourSeq 154 1366 1532 2000 95.2% chr19 - 38334530 38334694 165 browser details YourSeq 154 1371 1533 2000 97.6% chr4 + 123875915 123876088 174 browser details YourSeq 154 1371 1533 2000 96.3% chr13 + 3338603 3338763 161 browser details YourSeq 154 1369 1533 2000 97.0% chr12 + 71069282 71069450 169 browser details YourSeq 154 1365 1533 2000 94.0% chr1 + 128426145 128426310 166 browser details YourSeq 153 1371 1533 2000 98.2% chr18 - 84923658 84923822 165 browser details YourSeq 153 1371 1533 2000 97.0% chr17 - 28551897 28552059 163

Note: The 2000 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 1222 1 1222 1222 100.0% chr13 + 3642254 3643475 1222 browser details YourSeq 22 31 52 1222 100.0% chr9 - 44249210 44249231 22 browser details YourSeq 21 1200 1220 1222 100.0% chr4 - 131261361 131261381 21 browser details YourSeq 21 732 752 1222 100.0% chr2 - 18582696 18582716 21 browser details YourSeq 20 1106 1125 1222 100.0% chr10 - 75358188 75358207 20 browser details YourSeq 20 923 942 1222 100.0% chr1 - 73772409 73772428 20 browser details YourSeq 20 261 280 1222 100.0% chr1 + 182685887 182685906 20

Note: The 1222 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Asb13 ankyrin repeat and SOCS box-containing 13 [ Mus musculus (house mouse) ] Gene ID: 142688, updated on 12-Aug-2019

Gene summary

Official Symbol Asb13 provided by MGI Official Full Name ankyrin repeat and SOCS box-containing 13 provided by MGI Primary source MGI:MGI:2145525 See related Ensembl:ENSMUSG00000033781 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 Asb-13; C85285; 2210015B19Rik; 6430573K02Rik Expression Ubiquitous expression in kidney adult (RPKM 25.8), liver adult (RPKM 18.0) and 27 other tissuesS ee more Orthologs human all

Genomic context

Location: 13; 13 A1 See Asb13 in Genome Data Viewer Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (3633589..3653822)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (3633322..3651025)

Chromosome 13 - NC_000079.6

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Transcript information: This gene has 4 transcripts

Gene: Asb13 ENSMUSG00000033781

Description ankyrin repeat and SOCS box-containing 13 [Source:MGI Symbol;Acc:MGI:2145525] Gene Synonyms 2210015B19Rik, 6430573K02Rik Location Chromosome 13: 3,634,032-3,653,822 forward strand. GRCm38:CM001006.2 About this gene This gene has 4 transcripts (splice variants), 207 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Asb13-201 ENSMUST00000042288.7 4453 278aa ENSMUSP00000046476.6 Protein coding CCDS36582 Q8VBX0 TSL:1 GENCODE basic APPRIS P1

Asb13-203 ENSMUST00000141967.1 838 No protein - Retained intron - - TSL:3

Asb13-202 ENSMUST00000141147.1 576 No protein - lncRNA - - TSL:3

Asb13-204 ENSMUST00000156595.1 456 No protein - lncRNA - - TSL:1

39.79 kb Forward strand 3.63Mb 3.64Mb 3.65Mb 3.66Mb Genes (Comprehensive set... Asb13-201 >protein coding

Asb13-204 >lncRNA Gm23084-201 >rRNA

Asb13-202 >lncRNA

Asb13-203 >retained intron

Contigs < AC129597.5 Regulatory Build

3.63Mb 3.64Mb 3.65Mb 3.66Mb Reverse strand 39.79 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000042288

19.79 kb Forward strand

Asb13-201 >protein coding

ENSMUSP00000046... Low complexity (Seg) Superfamily Ankyrin repeat-containing domain superfamily SOCS box-like domain superfamily

SMART Ankyrin repeat SOCS box domain Prints Ankyrin repeat Pfam Ankyrin repeat-containing domain SOCS box domain PROSITE profiles Ankyrin repeat SOCS box domain

Ankyrin repeat-containing domain PANTHER PTHR24136

PTHR24136:SF31 Gene3D Ankyrin repeat-containing domain superfamily 1.10.750.20

CDD Ankyrin repeat and SOCS box protein 13, SOCS box domain

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

Variant Legend splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 278

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

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