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Mouse Cox6b2 Knockout Project (CRISPR/Cas9)

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Mouse Cox6b2 Knockout Project (CRISPR/Cas9) http://www.alphaknockout.com/ Mouse Cox6b2 Knockout Project (CRISPR/Cas9) Objective: To create a Cox6b2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cox6b2 gene (NCBI Reference Sequence: NM_183405 ; Ensembl: ENSMUSG00000051811 ) is located on Mouse chromosome 7. 4 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000063324). Exon 3 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 42.8% of the coding region. Exon 3 covers 38.26% of the coding region. The size of effective KO region: ~101 bp. The KO region does not have any other known gene. Page 1 of 9 http://www.alphaknockout.com/ Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 Legends Exon of mouse Cox6b2 Knockout region Page 2 of 9 http://www.alphaknockout.com/ Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 562 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 85 bp section downstream 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. Page 3 of 9 http://www.alphaknockout.com/ Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(562bp) | A(23.31% 131) | C(18.68% 105) | G(41.81% 235) | T(16.19% 91) Note: The 562 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(85bp) | A(17.65% 15) | C(21.18% 18) | G(43.53% 37) | T(17.65% 15) Note: The 85 bp section downstream 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. Page 4 of 9 http://www.alphaknockout.com/ BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 562 1 562 562 100.0% chr7 - 4752162 4752723 562 browser details YourSeq 25 311 342 562 96.3% chr12 + 86859881 86859915 35 browser details YourSeq 24 84 123 562 65.4% chr17 - 83547545 83547571 27 browser details YourSeq 23 284 310 562 79.2% chr14 - 43917810 43917833 24 browser details YourSeq 23 455 478 562 100.0% chr10 - 128770134 128770159 26 browser details YourSeq 22 130 155 562 92.4% chr8 - 15135416 15135441 26 browser details YourSeq 21 223 243 562 100.0% chr1 + 158258843 158258863 21 browser details YourSeq 21 149 169 562 100.0% chr1 + 125341103 125341123 21 browser details YourSeq 20 313 332 562 100.0% chr11 - 96049565 96049584 20 Note: The 562 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 85 1 85 85 100.0% chr7 - 4751976 4752060 85 browser details YourSeq 20 21 40 85 100.0% chr4 + 45736981 45737000 20 Note: The 85 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 http://www.alphaknockout.com/ Gene and protein information: Cox6b2 cytochrome c oxidase subunit 6B2 [ Mus musculus (house mouse) ] Gene ID: 333182, updated on 12-Aug-2019 Gene summary Official Symbol Cox6b2 provided by MGI Official Full Name cytochrome c oxidase subunit 6B2 provided by MGI Primary source MGI:MGI:3044182 See related Ensembl:ENSMUSG00000051811 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 COXVIB2; BC048670; 1700067P11Rik Expression Biased expression in testis adult (RPKM 381.6), liver E14.5 (RPKM 28.1) and 1 other tissueS ee more Orthologs human all Genomic context Location: 7; 7 A1 See Cox6b2 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (4751792..4753094, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (4703396..4704696, complement) Chromosome 7 - NC_000073.6 Page 6 of 9 http://www.alphaknockout.com/ Transcript information: This gene has 9 transcripts Gene: Cox6b2 ENSMUSG00000051811 Description cytochrome c oxidase subunit 6B2 [Source:MGI Symbol;Acc:MGI:3044182] Gene Synonyms 1700067P11Rik, COXVIB2 Location Chromosome 7: 4,751,792-4,753,094 reverse strand. GRCm38:CM001000.2 About this gene This gene has 9 transcripts (splice variants), 242 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cox6b2- ENSMUST00000182111.7 585 88aa ENSMUSP00000138709.1 Protein coding CCDS39738 Q059Q8 TSL:1 203 Q80ZN9 GENCODE basic APPRIS P1 Cox6b2- ENSMUST00000063324.13 553 88aa ENSMUSP00000064988.6 Protein coding CCDS39738 Q059Q8 TSL:1 201 Q80ZN9 GENCODE basic APPRIS P1 Cox6b2- ENSMUST00000182048.1 465 88aa ENSMUSP00000138765.1 Protein coding CCDS39738 Q059Q8 TSL:1 202 Q80ZN9 GENCODE basic APPRIS P1 Cox6b2- ENSMUST00000184143.7 435 60aa ENSMUSP00000139239.1 Protein coding CCDS80658 V9GXN3 TSL:2 209 GENCODE basic Cox6b2- ENSMUST00000182738.7 423 88aa ENSMUSP00000138744.1 Protein coding CCDS80657 S4R2Q6 TSL:2 206 GENCODE basic Cox6b2- ENSMUST00000183971.7 401 78aa ENSMUSP00000138911.1 Protein coding - V9GWZ7 TSL:5 208 GENCODE basic Cox6b2- ENSMUST00000182173.1 391 104aa ENSMUSP00000138288.1 Protein coding - S4R1N0 TSL:2 204 GENCODE basic Cox6b2- ENSMUST00000183334.7 490 46aa ENSMUSP00000138708.1 Nonsense mediated - S4R2M9 TSL:3 207 decay Cox6b2- ENSMUST00000182272.1 519 No - Retained intron - - TSL:2 205 protein Page 7 of 9 http://www.alphaknockout.com/ 21.30 kb Forward strand 4.745Mb 4.750Mb 4.755Mb 4.760Mb Genes Kmt5c-201 >protein coding (Comprehensive set... Kmt5c-208 >retained intron Kmt5c-204 >protein coding Kmt5c-207 >protein coding Kmt5c-202 >protein coding Kmt5c-203 >protein coding Kmt5c-205 >retained intron Kmt5c-212 >nonsense mediated decay Kmt5c-209 >retained intron Kmt5c-211 >retained intron Kmt5c-206 >retained intron Kmt5c-210 >retained intron Contigs < AC161197.9 Genes < Cox6b2-201protein coding (Comprehensive set... < Cox6b2-209protein coding < Cox6b2-203protein coding < Cox6b2-208protein coding < Cox6b2-206protein coding < Cox6b2-207nonsense mediated decay < Cox6b2-202protein coding < Cox6b2-205retained intron < Cox6b2-204protein coding < Fam71e2-201nonsense mediated decay < Fam71e2-202protein coding Regulatory Build 4.745Mb 4.750Mb 4.755Mb 4.760Mb Reverse strand 21.30 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 8 of 9 http://www.alphaknockout.com/ Transcript: ENSMUST00000063324 < Cox6b2-201protein coding Reverse strand 1.30 kb ENSMUSP00000064... MobiDB lite Superfamily Cytochrome c oxidase, subunit VIb superfamily Pfam Cytochrome c oxidase, subunit VIb PROSITE profiles PS51808 PIRSF Cytochrome c oxidase, subunit VIb PANTHER Cytochrome c oxidase, subunit VIb PTHR11387:SF12 Gene3D Cytochrome c oxidase, subunit VIb superfamily CDD Cytochrome c oxidase, subunit VIb All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Y Variant Legend missense variant Scale bar 0 8 16 24 32 40 48 56 64 72 80 88 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC, VectorBuilder. Page 9 of 9.
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