https://www.alphaknockout.com

Mouse Kank1 Knockout Project (CRISPR/Cas9)

Objective: To create a Kank1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Kank1 (NCBI Reference Sequence: NM_181404 ; Ensembl: ENSMUSG00000032702 ) is located on Mouse 19. 14 exons are identified, with the ATG start codon in exon 4 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000049400). Exon 5 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 5 starts from about 0.93% of the coding region. Exon 5 covers 65.66% of the coding region. The size of effective KO region: ~2679 bp. The KO 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 14

Legends Exon of mouse Kank1 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 of Exon 5 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 2000 bp section of Exon 5 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(26.55% 531) | C(26.35% 527) | T(18.3% 366) | G(28.8% 576)

Note: The 2000 bp section of Exon 5 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(2000bp) | A(25.35% 507) | C(25.1% 502) | T(18.15% 363) | G(31.4% 628)

Note: The 2000 bp section of Exon 5 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% chr19 + 25409088 25411087 2000 browser details YourSeq 23 1758 1783 2000 96.0% chr1 + 126486775 126486811 37 browser details YourSeq 22 794 817 2000 95.9% chr11 - 45647064 45647087 24 browser details YourSeq 21 1535 1557 2000 95.7% chr10 + 41110149 41110171 23 browser details YourSeq 20 1355 1376 2000 95.5% chr11 + 76143736 76143757 22

Note: The 2000 bp section 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 2000 1 2000 2000 100.0% chr19 + 25409765 25411764 2000 browser details YourSeq 36 1358 1422 2000 95.2% chr16 - 17530101 17530167 67 browser details YourSeq 25 1592 1618 2000 96.3% chr10 - 89034084 89034110 27 browser details YourSeq 23 1081 1106 2000 96.0% chr1 + 126486775 126486811 37 browser details YourSeq 22 117 140 2000 95.9% chr11 - 45647064 45647087 24 browser details YourSeq 21 858 880 2000 95.7% chr10 + 41110149 41110171 23 browser details YourSeq 20 1752 1771 2000 100.0% chr1 - 77655313 77655332 20 browser details YourSeq 20 678 699 2000 95.5% chr11 + 76143736 76143757 22

Note: The 2000 bp section of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Kank1 KN motif and repeat domains 1 [ Mus musculus (house mouse) ] Gene ID: 107351, updated on 12-Aug-2019

Gene summary

Official Symbol Kank1 provided by MGI Official Full Name KN motif and ankyrin repeat domains 1 provided by MGI Primary source MGI:MGI:2147707 See related Ensembl:ENSMUSG00000032702 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 Ankrd15; AU015049; AW121052; mKIAA0172; A930031B09Rik; D330024H06Rik Expression Ubiquitous expression in bladder adult (RPKM 22.5), ovary adult (RPKM 15.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 19; 19 B See Kank1 in Genome Data Viewer Exon count: 20

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (25236732..25434498)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (25311692..25508986)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 4 transcripts

Gene: Kank1 ENSMUSG00000032702

Description KN motif and ankyrin repeat domains 1 [Source:MGI Symbol;Acc:MGI:2147707] Gene Synonyms A930031B09Rik, Ankrd15, D330024H06Rik Location Chromosome 19: 25,236,975-25,434,496 forward strand. GRCm38:CM001012.2 About this gene This gene has 4 transcripts (splice variants), 278 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Kank1-201 ENSMUST00000049400.14 5637 1360aa ENSMUSP00000042177.8 Protein coding CCDS37944 E9Q238 TSL:5 GENCODE basic APPRIS P1

Kank1-203 ENSMUST00000146647.2 3655 1147aa ENSMUSP00000116660.1 Protein coding - E9Q944 CDS 3' incomplete TSL:1

Kank1-204 ENSMUST00000155788.1 583 No protein - Retained intron - - TSL:2

Kank1-202 ENSMUST00000137260.1 756 No protein - lncRNA - - TSL:3

217.52 kb Forward strand 25.25Mb 25.30Mb 25.35Mb 25.40Mb (Comprehensive set... Kank1-201 >protein coding

Kank1-202 >lncRNA

Kank1-203 >protein coding

Kank1-204 >retained intron

Contigs < AC132319.3 < AC126944.3

Genes < Gm34432-201processed pseudogene (Comprehensive set...

Regulatory Build

25.25Mb 25.30Mb 25.35Mb 25.40Mb Reverse strand 217.52 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript pseudogene RNA gene

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Transcript: ENSMUST00000049400

197.52 kb Forward strand

Kank1-201 >protein coding

ENSMUSP00000042... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Ankyrin repeat-containing domain superfamily SMART Ankyrin repeat Pfam Kank N-terminal motif Ankyrin repeat-containing domain

PROSITE profiles Ankyrin repeat-containing domain

Ankyrin repeat PANTHER PTHR24168

PTHR24168:SF19 Gene3D Ankyrin repeat-containing domain superfamily

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

Variant Legend stop gained missense variant synonymous variant

Scale bar 0 200 400 600 800 1000 1360

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

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