https://www.alphaknockout.com

Mouse Atp6v0b Knockout Project (CRISPR/Cas9)

Objective: To create a Atp6v0b knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Atp6v0b gene (NCBI Reference Sequence: NM_033617.3 ; Ensembl: ENSMUSG00000033379 ) is located on Mouse chromosome 4. 8 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000036380). Exon 3~6 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:

Exon 3 starts from about 19.02% of the coding region. Exon 3~6 covers 46.18% of the coding region. The size of effective KO region: ~731 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 3 4 5 6 8

Legends Exon of mouse Atp6v0b Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 230 bp section upstream of Exon 3 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 334 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(230bp) | A(19.13% 44) | C(28.26% 65) | T(26.09% 60) | G(26.52% 61)

Note: The 230 bp section upstream of Exon 3 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(334bp) | A(22.16% 74) | C(32.63% 109) | T(22.46% 75) | G(22.75% 76)

Note: The 334 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 230 1 230 230 100.0% chr4 - 117886360 117886589 230 browser details YourSeq 28 30 58 230 100.0% chr1 + 131755250 131755498 249 browser details YourSeq 26 153 184 230 96.6% chr6 + 145135505 145135542 38 browser details YourSeq 24 94 118 230 100.0% chr3 - 32552013 32552056 44 browser details YourSeq 24 94 118 230 100.0% chr17 + 91320285 91320328 44 browser details YourSeq 23 26 51 230 96.2% chr13 - 52432428 52432456 29 browser details YourSeq 23 51 74 230 100.0% chr12 - 108117998 108118022 25 browser details YourSeq 23 73 97 230 87.5% chr11 - 71685031 71685054 24 browser details YourSeq 20 128 147 230 100.0% chr3 + 121685472 121685491 20

Note: The 230 bp section upstream of Exon 3 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 334 1 334 334 100.0% chr4 - 117885295 117885628 334 browser details YourSeq 26 89 123 334 70.4% chr10 - 61363979 61364005 27 browser details YourSeq 25 80 109 334 88.9% chr1 - 180649046 180649074 29 browser details YourSeq 24 89 112 334 100.0% chr17 - 33637034 33637057 24 browser details YourSeq 24 192 218 334 96.3% chr1 + 144169811 144169841 31 browser details YourSeq 22 236 258 334 100.0% chr10 - 79116505 79116528 24 browser details YourSeq 22 195 218 334 95.9% chr1 - 147836590 147836613 24 browser details YourSeq 21 198 218 334 100.0% chr8 + 7620486 7620506 21 browser details YourSeq 20 197 218 334 95.5% chr10 - 124470371 124470392 22 browser details YourSeq 20 195 218 334 91.7% chr1 - 96009171 96009194 24

Note: The 334 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Atp6v0b ATPase, H+ transporting, lysosomal V0 subunit B [ Mus musculus (house mouse) ] Gene ID: 114143, updated on 26-Jun-2020

Gene summary

Official Symbol Atp6v0b provided by MGI Official Full Name ATPase, H+ transporting, lysosomal V0 subunit B provided by MGI Primary source MGI:MGI:1890510 See related Ensembl:ENSMUSG00000033379 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Atp6f; VMA16; 2310024H13Rik Expression Ubiquitous expression in kidney adult (RPKM 210.8), adrenal adult (RPKM 164.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 D2.1 See Atp6v0b in Genome Data Viewer Exon count: 8

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (117884330..117887333, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (117556935..117559934, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 9 transcripts

Gene: Atp6v0b ENSMUSG00000033379

Description ATPase, H+ transporting, lysosomal V0 subunit B [Source:MGI Symbol;Acc:MGI:1890510] Gene Synonyms 2310024H13Rik, Atp6f, VMA16 Location Chromosome 4: 117,884,326-117,887,333 reverse strand. GRCm38:CM000997.2 About this gene This gene has 9 transcripts (splice variants), 270 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Atp6v0b- ENSMUST00000036380.13 1000 205aa ENSMUSP00000047682.7 Protein coding CCDS18540 Q3U889 TSL:1 201 Q91V37 GENCODE basic APPRIS P1

Atp6v0b- ENSMUST00000150204.7 935 158aa ENSMUSP00000119988.2 Protein coding - A0A0A0MQJ5 TSL:5 208 GENCODE basic

Atp6v0b- ENSMUST00000132073.1 872 84aa ENSMUSP00000137654.1 Protein coding - M0QW50 CDS 3' 203 incomplete TSL:5

Atp6v0b- ENSMUST00000147845.1 776 4aa ENSMUSP00000137945.1 Protein coding - - CDS 3' 206 incomplete TSL:2

Atp6v0b- ENSMUST00000149868.7 547 107aa ENSMUSP00000137788.1 Protein coding - M0QWE4 CDS 3' 207 incomplete TSL:3

Atp6v0b- ENSMUST00000136596.1 348 18aa ENSMUSP00000118538.2 Protein coding - A0A0A0MQI2 CDS 3' 205 incomplete TSL:3

Atp6v0b- ENSMUST00000124974.1 779 No - Retained - - TSL:5 202 protein intron

Atp6v0b- ENSMUST00000133530.7 646 No - Retained - - TSL:2 204 protein intron

Atp6v0b- ENSMUST00000152356.1 560 No - Retained - - TSL:2 209 protein intron

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23.01 kb Forward strand 117.875Mb 117.880Mb 117.885Mb 117.890Mb 117.895Mb Genes Slc6a9-207 >nonsense mediated decay Gm12841-201 >antisense (Comprehensive set...

Slc6a9-212 >retained intron

Contigs AL627128.16 > Genes < B4galt2-202nonsense mediated decay < Atp6v0b-201protein coding < Dph2-201protein coding < Ipo13-202processed transcript (Comprehensive set...

< B4galt2-206nonsense mediated decay < Atp6v0b-204retained intron < Dph2-203retained intron < Ipo13-206processed transcript

< B4galt2-201protein coding < Atp6v0b-208protein coding < Dph2-204nonsense mediated decay < Ipo13-201protein coding

< B4galt2-203protein coding < Atp6v0b-209retained intron < Dph2-202retained intron

< B4galt2-205protein coding < Atp6v0b-202retained intron < Ipo13-204processed transcript

< B4galt2-204processed transcript < Atp6v0b-203protein coding < Ipo13-203processed transcript

< B4galt2-207protein coding < Atp6v0b-207protein coding

< Atp6v0b-205protein coding

< Atp6v0b-206protein coding

Regulatory Build

117.875Mb 117.880Mb 117.885Mb 117.890Mb 117.895Mb Reverse strand 23.01 kb

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

Regulation Legend Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

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Transcript: ENSMUST00000036380

< Atp6v0b-201protein coding

Reverse strand 3.00 kb

ENSMUSP00000047... Transmembrane heli... Low complexity (Seg) Superfamily F/V-ATP synthase subunit C superfamily Prints V-ATPase proteolipid subunit Pfam V-ATPase proteolipid subunit C-like domain PANTHER PTHR10263:SF18

PTHR10263 Gene3D 1.20.120.610 CDD cd18177 cd18178

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 205

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

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