https://www.alphaknockout.com

Mouse Bbs9 Knockout Project (CRISPR/Cas9)

Objective: To create a Bbs9 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Bbs9 gene (NCBI Reference Sequence: NM_178415 ; Ensembl: ENSMUSG00000035919 ) is located on Mouse chromosome 9. 23 exons are identified, with the ATG start codon in exon 3 and the TAG stop codon in exon 23 (Transcript: ENSMUST00000150395). Exon 3~4 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: Mice homozygous for an ENU-induced allele exhibit abnormal pelvic girdle bone morphology, short tibia, increased body weight and increased body fat mass.

Exon 3 starts from the coding region. Exon 3~4 covers 9.96% of the coding region. The size of effective KO region: ~6042 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 23

Legends Exon of mouse Bbs9 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 3 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 4 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(20.7% 414) | C(22.2% 444) | T(36.15% 723) | G(20.95% 419)

Note: The 2000 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(2000bp) | A(24.65% 493) | C(22.3% 446) | T(31.55% 631) | G(21.5% 430)

Note: The 2000 bp section downstream of Exon 4 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% chr9 + 22488830 22490829 2000 browser details YourSeq 71 670 899 2000 75.6% chr3 + 108609014 108609199 186 browser details YourSeq 69 656 899 2000 74.0% chr12 - 58643744 58643939 196 browser details YourSeq 65 1537 1665 2000 88.3% chr6 - 146298874 146299017 144 browser details YourSeq 54 1572 1710 2000 84.7% chr1 - 131597090 131597236 147 browser details YourSeq 53 1616 1711 2000 85.4% chr5 + 146631604 146631701 98 browser details YourSeq 53 780 837 2000 96.6% chr12 + 74012191 74012252 62 browser details YourSeq 52 1568 1665 2000 92.1% chr1 + 128546145 128546465 321 browser details YourSeq 51 780 830 2000 100.0% chr7 - 18512935 18512985 51 browser details YourSeq 49 1618 1711 2000 73.8% chr4 + 42206944 42207031 88 browser details YourSeq 49 1558 1661 2000 94.7% chr1 + 193377519 193377633 115 browser details YourSeq 48 780 839 2000 92.9% chr12 + 74012131 74012198 68 browser details YourSeq 47 1618 1709 2000 81.4% chr1 - 37654125 37654216 92 browser details YourSeq 47 652 709 2000 91.4% chr15 + 6220501 6220559 59 browser details YourSeq 46 784 839 2000 92.8% chr1 - 120319306 120319369 64 browser details YourSeq 46 664 899 2000 68.2% chr11 + 40824994 40825184 191 browser details YourSeq 45 646 708 2000 94.4% chr15 - 96190936 96191004 69 browser details YourSeq 45 1617 1681 2000 91.0% chr3 + 103432033 103432238 206 browser details YourSeq 44 654 708 2000 91.0% chr15 - 82313059 82313114 56 browser details YourSeq 44 1568 1661 2000 71.0% chr1 + 149928438 149928512 75

Note: The 2000 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 2000 1 2000 2000 100.0% chr9 + 22496861 22498860 2000 browser details YourSeq 177 1722 2000 2000 95.0% chr3 + 13672730 13673603 874 browser details YourSeq 177 1801 2000 2000 94.9% chr14 + 31065907 31066105 199 browser details YourSeq 176 1445 1991 2000 86.4% chr2 - 167915486 167915820 335 browser details YourSeq 174 1804 2000 2000 94.9% chr10 - 31716492 31716693 202 browser details YourSeq 172 1816 2000 2000 96.8% chr1 - 175139407 175139594 188 browser details YourSeq 172 1813 2000 2000 95.8% chr19 + 5794382 5794569 188 browser details YourSeq 171 1815 1995 2000 97.8% chr2 - 126166114 126166298 185 browser details YourSeq 171 1815 2000 2000 96.8% chr5 + 34035479 34037159 1681 browser details YourSeq 171 1808 2000 2000 95.3% chr4 + 128928443 128929058 616 browser details YourSeq 171 1807 1991 2000 96.8% chr4 + 40369891 40370081 191 browser details YourSeq 170 1816 2000 2000 96.3% chr9 - 106254937 106255124 188 browser details YourSeq 170 1795 1995 2000 92.6% chr11 - 99899609 99899803 195 browser details YourSeq 170 1816 2000 2000 96.2% chr1 - 71131063 71131249 187 browser details YourSeq 170 1816 2000 2000 96.2% chr15 + 89768324 89768510 187 browser details YourSeq 170 1816 2000 2000 96.2% chr1 + 30676338 30676524 187 browser details YourSeq 170 1814 2000 2000 96.3% chr1 + 18006571 18007250 680 browser details YourSeq 169 1816 2000 2000 96.2% chr3 - 133592605 133592880 276 browser details YourSeq 169 1816 1995 2000 97.3% chr3 - 73486859 73487041 183 browser details YourSeq 169 1817 2000 2000 96.2% chr2 - 28647034 28647219 186

Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Bbs9 Bardet-Biedl syndrome 9 (human) [ Mus musculus (house mouse) ] Gene ID: 319845, updated on 24-Oct-2019

Gene summary

Official Symbol Bbs9 provided by MGI Official Full Name Bardet-Biedl syndrome 9 (human) provided by MGI Primary source MGI:MGI:2442833 See related Ensembl:ENSMUSG00000035919 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 E130103I17Rik Expression Ubiquitous expression in testis adult (RPKM 9.5), CNS E18 (RPKM 5.2) and 26 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 A3 See Bbs9 in Genome Data Viewer Exon count: 26

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (22475559..22888283)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (22280159..22692724)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 12 transcripts

Gene: Bbs9 ENSMUSG00000035919

Description Bardet-Biedl syndrome 9 (human) [Source:MGI Symbol;Acc:MGI:2442833] Gene Synonyms E130103I17Rik, EST 3159894 Location Chromosome 9: 22,475,715-22,888,280 forward strand. GRCm38:CM001002.2 About this gene This gene has 12 transcripts (splice variants), 204 orthologues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Bbs9- ENSMUST00000147712.7 3495 880aa ENSMUSP00000122058.1 Protein coding CCDS22928 Q811G0 TSL:1 210 GENCODE basic APPRIS P2

Bbs9- ENSMUST00000150395.7 3425 880aa ENSMUSP00000116629.1 Protein coding CCDS22928 Q811G0 TSL:1 211 GENCODE basic APPRIS P2

Bbs9- ENSMUST00000039798.15 3382 880aa ENSMUSP00000043042.9 Protein coding CCDS22928 Q811G0 TSL:1 201 GENCODE basic APPRIS P2

Bbs9- ENSMUST00000147405.7 3045 885aa ENSMUSP00000120927.1 Protein coding - Q811G0 TSL:5 209 GENCODE basic APPRIS ALT1

Bbs9- ENSMUST00000128812.7 833 147aa ENSMUSP00000117700.1 Protein coding - D3Z389 CDS 3' 204 incomplete TSL:5

Bbs9- ENSMUST00000142313.7 833 132aa ENSMUSP00000122325.1 Protein coding - D3Z6T1 CDS 3' 208 incomplete TSL:3

Bbs9- ENSMUST00000136084.7 638 163aa ENSMUSP00000123160.1 Protein coding - F6XSQ3 CDS 5' 206 incomplete TSL:5

Bbs9- ENSMUST00000127296.7 1640 61aa ENSMUSP00000121992.1 Nonsense mediated - D6RIM6 CDS 5' 203 decay incomplete TSL:5

Bbs9- ENSMUST00000130479.1 2641 No - Retained intron - - TSL:1 205 protein

Bbs9- ENSMUST00000124076.1 1012 No - Retained intron - - TSL:3 202 protein

Bbs9- ENSMUST00000152719.7 716 No - lncRNA - - TSL:5 212 protein

Bbs9- ENSMUST00000137547.1 542 No - lncRNA - - TSL:3 207 protein

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432.57 kb Forward strand 22.5Mb 22.6Mb 22.7Mb 22.8Mb Genes (Comprehensive set... Bbs9-201 >protein coding

Bbs9-211 >protein coding

Bbs9-210 >protein coding

Bbs9-208 >protein coding Bbs9-207 >lncRNA Bbs9-206 >protein coding

Bbs9-204 >protein coding Bbs9-203 >nonsense mediated decay

Bbs9-209 >protein coding

Bbs9-212 >lncRNA Bbs9-205 >retained intron

Bbs9-202 >retained intron

Contigs < AC113106.9 AC113127.6 > < AC164882.2 Genes < Rp9-203protein coding < Gm16165-201processed pseudogene < Gm27639-201snRNA (Comprehensive set...

< Rp9-201protein coding < Gm16164-201processed pseudogene

< Rp9-204protein coding

< Rp9-205retained intron

< Rp9-202retained intron

< Gm2976-201processed pseudogene

Regulatory Build

22.5Mb 22.6Mb 22.7Mb 22.8Mb Reverse strand 432.57 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

pseudogene RNA gene processed transcript

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Transcript: ENSMUST00000150395

412.55 kb Forward strand

Bbs9-211 >protein coding

ENSMUSP00000116... Low complexity (Seg) Coiled-coils (Ncoils) Pfam PTHB1, N-terminal domain PTHB1, C-terminal domain

PANTHER Parathyroid hormone-responsive B1

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

Variant Legend

missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 880

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

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