https://www.alphaknockout.com

Mouse Kdm1b Knockout Project (CRISPR/Cas9)

Objective: To create a Kdm1b knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Kdm1b (NCBI Reference Sequence: NM_172262 ; Ensembl: ENSMUSG00000038080 ) is located on Mouse 13. 21 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 21 (Transcript: ENSMUST00000037025). Exon 2~6 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: Homozygous null mice of both sexes are viable, grossly normal and male mice are fertile; however, heterozygous progeny of homozygous null mothers display severe placental defects, embryonic growth impairment, neural tube defects and pericardial edema, and do not survive past E10.5.

Exon 2 starts from the coding region. Exon 2~6 covers 21.55% of the coding region. The size of effective KO region: ~5833 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 2 3 4 5 6 21

Legends Exon of mouse Kdm1b 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 2000 bp section downstream of Exon 6 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(25.7% 514) | C(19.15% 383) | T(32.45% 649) | G(22.7% 454)

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(2000bp) | A(26.3% 526) | C(19.0% 380) | T(30.2% 604) | G(24.5% 490)

Note: The 2000 bp section downstream of Exon 6 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 + 47045917 47047916 2000 browser details YourSeq 52 670 740 2000 92.1% chr3 + 104368905 104368977 73 browser details YourSeq 52 642 782 2000 67.8% chr14 + 33780224 33780345 122 browser details YourSeq 47 663 782 2000 79.3% chr15 - 99934448 99934552 105 browser details YourSeq 43 670 780 2000 75.6% chr18 - 56585849 56585942 94 browser details YourSeq 43 687 782 2000 90.6% chr1 + 133334304 133334401 98 browser details YourSeq 42 663 721 2000 86.6% chr12 - 85422837 85422894 58 browser details YourSeq 42 1736 1791 2000 83.7% chr2 + 163607815 163607869 55 browser details YourSeq 41 694 743 2000 93.8% chrX + 85855705 85855755 51 browser details YourSeq 41 663 745 2000 91.7% chr17 + 70811213 70811679 467 browser details YourSeq 37 1334 1764 2000 51.2% chr2 + 21241080 21241143 64 browser details YourSeq 36 670 728 2000 87.8% chr2 - 32604295 32604353 59 browser details YourSeq 36 665 722 2000 83.4% chr13 + 54778755 54778811 57 browser details YourSeq 34 695 782 2000 92.5% chr18 + 56556812 56556901 90 browser details YourSeq 34 705 796 2000 85.8% chr15 + 8379791 8379881 91 browser details YourSeq 34 770 823 2000 75.6% chr1 + 186851429 186851479 51 browser details YourSeq 33 665 734 2000 87.2% chr19 - 36874144 36874212 69 browser details YourSeq 33 717 780 2000 94.6% chr10 - 25542706 25542769 64 browser details YourSeq 32 672 721 2000 86.9% chr2 + 102163580 102163628 49 browser details YourSeq 31 670 732 2000 74.7% chr19 - 10583460 10583522 63

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 2000 1 2000 2000 100.0% chr13 + 47053737 47055736 2000 browser details YourSeq 62 1031 1602 2000 66.7% chr16 + 5463426 5463569 144 browser details YourSeq 52 1455 1566 2000 76.5% chr5 + 52244508 52244606 99 browser details YourSeq 51 1513 1601 2000 93.3% chrX + 103982834 103983362 529 browser details YourSeq 47 1513 1597 2000 98.0% chr11 + 116357228 116357453 226 browser details YourSeq 44 1514 1601 2000 75.0% chr17 + 28521410 28521497 88 browser details YourSeq 43 1513 1601 2000 92.2% chr11 + 95155944 95156033 90 browser details YourSeq 40 1481 1601 2000 95.5% chr19 - 46873950 46874070 121 browser details YourSeq 39 1513 1566 2000 95.4% chr7 + 11189713 11189767 55 browser details YourSeq 38 1516 1601 2000 95.3% chr12 - 109083275 109083361 87 browser details YourSeq 38 1523 1601 2000 95.2% chr6 + 72941226 72941304 79 browser details YourSeq 38 1518 1600 2000 93.4% chr5 + 130686389 130686473 85 browser details YourSeq 38 1512 1564 2000 93.2% chr16 + 25864881 25864934 54 browser details YourSeq 37 1513 1566 2000 93.1% chr9 + 102614878 102614932 55 browser details YourSeq 37 1513 1566 2000 93.1% chr19 + 53916103 53916157 55 browser details YourSeq 36 1513 1597 2000 70.0% chr1 - 127859623 127859679 57 browser details YourSeq 35 1484 1566 2000 83.0% chr14 + 70592415 70592496 82 browser details YourSeq 34 1513 1566 2000 94.8% chr19 - 50456314 50456369 56 browser details YourSeq 33 1513 1566 2000 88.4% chr5 + 120512443 120512497 55 browser details YourSeq 33 1519 1601 2000 89.2% chr4 + 147982466 147982547 82

Note: The 2000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Kdm1b lysine (K)-specific 1B [ Mus musculus (house mouse) ] Gene ID: 218214, updated on 10-Oct-2019

Gene summary

Official Symbol Kdm1b provided by MGI Official Full Name lysine (K)-specific demethylase 1B provided by MGI Primary source MGI:MGI:2145261 See related Ensembl:ENSMUSG00000038080 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 Aof1; AI482520; 4632428N09Rik Expression Ubiquitous expression in bladder adult (RPKM 25.1), liver E14 (RPKM 13.2) and 27 other tissues See more Orthologs human all

Genomic context

Location: 13; 13 A5 See Kdm1b in Genome Data Viewer Exon count: 23

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (47043373..47085279)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (47138908..47179982)

Chromosome 13 - NC_000079.6

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Transcript information: This gene has 5 transcripts

Gene: Kdm1b ENSMUSG00000038080

Description lysine (K)-specific demethylase 1B [Source:MGI Symbol;Acc:MGI:2145261] Gene Synonyms 4632428N09Rik, Aof1 Location Chromosome 13: 47,043,499-47,085,279 forward strand. GRCm38:CM001006.2 About this gene This gene has 5 transcripts (splice variants), 205 orthologues, 9 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Kdm1b-201 ENSMUST00000037025.15 4987 826aa ENSMUSP00000038373.8 Protein coding CCDS26489 Q8CIG3 TSL:1 GENCODE basic APPRIS P1

Kdm1b-204 ENSMUST00000143518.2 2969 443aa ENSMUSP00000114999.2 Protein coding - F6V3V2 CDS 5' incomplete TSL:1

Kdm1b-205 ENSMUST00000143868.1 736 176aa ENSMUSP00000117793.1 Protein coding - D3Z353 CDS 3' incomplete TSL:3

Kdm1b-203 ENSMUST00000131120.7 870 No protein - Retained intron - - TSL:3

Kdm1b-202 ENSMUST00000128977.2 2876 No protein - lncRNA - - TSL:1

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61.78 kb Forward strand 47.04Mb 47.05Mb 47.06Mb 47.07Mb 47.08Mb 47.09Mb (Comprehensive set... Kdm1b-201 >protein coding

Kdm1b-205 >protein coding Kdm1b-204 >protein coding

Kdm1b-203 >retained intron

Kdm1b-202 >lncRNA

Contigs AC096628.14 > < AC154171.5 Genes < Tpmt-201protein coding < Dek-207nonsense mediated decay (Comprehensive set...

< Tpmt-202protein coding < Dek-204lncRNA

< Tpmt-206protein coding < Dek-201protein coding

< Tpmt-205protein coding < Dek-203protein coding

< Tpmt-207protein coding

< Tpmt-204retained intron

< Tpmt-203protein coding

Regulatory Build

47.04Mb 47.05Mb 47.06Mb 47.07Mb 47.08Mb 47.09Mb Reverse strand 61.78 kb

Regulation Legend CTCF Enhancer Open Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000037025

41.78 kb Forward strand

Kdm1b-201 >protein coding

ENSMUSP00000038... MobiDB lite Low complexity (Seg) Superfamily Homeobox-like domain superfamily SSF54373

FAD/NAD(P)-binding domain superfamily Prints PR00419 Pfam Zinc finger, CW-type SWIRM domain Amine oxidase

PROSITE profiles Zinc finger, CW-type SWIRM domain

PANTHER PTHR10742:SF359

PTHR10742 Gene3D 3.30.40.100 FAD/NAD(P)-binding domain superfamily

3.90.660.10

Winged helix-like DNA-binding domain superfamily

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

Variant Legend missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 826

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

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