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Mouse Sptbn2 Knockout Project (CRISPR/Cas9)

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Mouse Sptbn2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Sptbn2 Knockout Project (CRISPR/Cas9) Objective: To create a Sptbn2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Sptbn2 gene (NCBI Reference Sequence: NM_021287 ; Ensembl: ENSMUSG00000067889 ) is located on Mouse chromosome 19. 37 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 37 (Transcript: ENSMUST00000008991). Exon 3~12 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 hypomorphic mutants exhibit a progressive ataxic phenotype with gait abnormalities, tremor, deteriorating motor coordination, Purkinje cell loss, and cerebellar atrophy (molecular layer thinning) and age-related reduction in simple firing ratein surviving Purkinje cells. Exon 3 starts from about 2.21% of the coding region. Exon 3~12 covers 20.88% of the coding region. The size of effective KO region: ~8611 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 6 7 8 9 10 11 12 37 Legends Exon of mouse Sptbn2 Knockout region Page 2 of 9 https://www.alphaknockout.com 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 963 bp section downstream of Exon 12 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 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(20.1% 402) | C(25.95% 519) | T(30.85% 617) | G(23.1% 462) 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(963bp) | A(25.34% 244) | C(21.08% 203) | T(21.81% 210) | G(31.78% 306) Note: The 963 bp section downstream of Exon 12 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 https://www.alphaknockout.com 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 + 4722081 4724080 2000 browser details YourSeq 267 708 1591 2000 93.3% chr13 - 61922035 62095609 173575 browser details YourSeq 181 693 1503 2000 95.6% chr19 + 3800769 3955698 154930 browser details YourSeq 159 1443 1695 2000 94.4% chr19 + 23087362 23087990 629 browser details YourSeq 152 1424 1673 2000 88.2% chr4 + 141940291 141940491 201 browser details YourSeq 150 1426 1874 2000 95.8% chr1 + 86510629 86511248 620 browser details YourSeq 147 1417 1611 2000 88.9% chr14 - 113404863 113405418 556 browser details YourSeq 142 1437 1593 2000 95.6% chr11 + 29531765 29531931 167 browser details YourSeq 139 1430 1592 2000 93.8% chr10 + 45064822 45064994 173 browser details YourSeq 137 1428 1592 2000 93.1% chr17 - 62919236 62919417 182 browser details YourSeq 134 1423 1594 2000 94.8% chr11 - 106585191 106585370 180 browser details YourSeq 134 1449 1608 2000 93.0% chr5 + 33014746 33014916 171 browser details YourSeq 130 1433 1585 2000 94.1% chr1 + 46380115 46380298 184 browser details YourSeq 128 1442 1588 2000 93.9% chr10 - 21005740 21005896 157 browser details YourSeq 127 1451 1593 2000 95.2% chr5 - 36786669 36786851 183 browser details YourSeq 125 1427 1593 2000 93.3% chr14 - 111567810 111568001 192 browser details YourSeq 125 708 935 2000 93.8% chr1 - 16000373 16000766 394 browser details YourSeq 125 1436 1592 2000 94.4% chr9 + 22570912 22571105 194 browser details YourSeq 124 1450 1594 2000 93.7% chr4 - 115778320 115778471 152 browser details YourSeq 124 1448 1606 2000 92.0% chr14 - 8210720 8210962 243 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 963 1 963 963 100.0% chr19 + 4732692 4733654 963 browser details YourSeq 33 28 187 963 94.6% chr13 + 55842010 55842218 209 browser details YourSeq 25 675 700 963 100.0% chr1 + 133186176 133186679 504 browser details YourSeq 21 528 548 963 100.0% chr7 - 19762056 19762076 21 browser details YourSeq 21 501 521 963 100.0% chr14 + 77366493 77366513 21 browser details YourSeq 20 780 799 963 100.0% chr11 - 8635053 8635072 20 browser details YourSeq 20 172 191 963 100.0% chr1 + 154539077 154539096 20 Note: The 963 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Sptbn2 spectrin beta, non-erythrocytic 2 [ Mus musculus (house mouse) ] Gene ID: 20743, updated on 12-Aug-2019 Gene summary Official Symbol Sptbn2 provided by MGI Official Full Name spectrin beta, non-erythrocytic 2 provided by MGI Primary source MGI:MGI:1313261 See related Ensembl:ENSMUSG00000067889 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 Spnb3 Expression Biased expression in cortex adult (RPKM 49.2), frontal lobe adult (RPKM 46.9) and 7 other tissues See more Orthologs human all Genomic context Location: 19 A; 19 4.1 cM See Sptbn2 in Genome Data Viewer Exon count: 39 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (4711208..4753978) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (4711223..4752352) Chromosome 19 - NC_000085.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Sptbn2 ENSMUSG00000067889 Description spectrin beta, non-erythrocytic 2 [Source:MGI Symbol;Acc:MGI:1313261] Gene Synonyms Spnb3 Location Chromosome 19: 4,711,167-4,752,360 forward strand. GRCm38:CM001012.2 About this gene This gene has 4 transcripts (splice variants), 195 orthologues, 36 paralogues, is a member of 1 Ensembl protein family and is associated with 16 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Sptbn2-201 ENSMUST00000008991.7 8299 2388aa ENSMUSP00000008991.6 Protein coding CCDS29434 Q68FG2 TSL:1 GENCODE basic APPRIS P1 Sptbn2-202 ENSMUST00000237694.1 626 175aa ENSMUSP00000158522.1 Protein coding - A0A494BBH5 CDS 3' incomplete Sptbn2-204 ENSMUST00000238976.1 606 110aa ENSMUSP00000159038.1 Protein coding - - CDS 3' incomplete Sptbn2-203 ENSMUST00000238162.1 360 No protein - lncRNA - - - Page 7 of 9 https://www.alphaknockout.com 61.19 kb Forward strand 4.71Mb 4.72Mb 4.73Mb 4.74Mb 4.75Mb 4.76Mb Genes Sptbn2-201 >protein coding Rbm4b-205 >retained intron (Comprehensive set... Sptbn2-202 >protein coding Sptbn2-203 >lncRNA Rbm4b-207 >nonsense mediated decay Sptbn2-204 >protein coding Rbm4b-208 >nonsense mediated decay Rbm4b-209 >nonsense mediated decay Rbm4b-203 >nonsense mediated decay Rbm4b-202 >retained intron Rbm4b-206 >retained intron Rbm4b-204 >protein coding Rbm4b-201 >protein coding Rbm4b-210 >protein coding Contigs < AC147618.3 Genes < Gm21992-203nonsense mediated decay (Comprehensive set... < Gm37206-201TEC Regulatory Build 4.71Mb 4.72Mb 4.73Mb 4.74Mb 4.75Mb 4.76Mb Reverse strand 61.19 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000008991 41.19 kb Forward strand Sptbn2-201 >protein coding ENSMUSP00000008... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF46966 SSF50729 CH domain superfamily SMART Calponin homology domain Pleckstrin homology domain Spectrin/alpha-actinin Prints Pleckstrin homology domain, spectrin-type Pfam Calponin homology domain Pleckstrin homology domain 9 Spectrin repeat PROSITE profiles Calponin homology domain Pleckstrin homology domain PROSITE patterns Actinin-type actin-binding domain, conserved site Actinin-type actin-binding domain, conserved site PIRSF Spectrin, beta subunit PANTHER PTHR11915 PTHR11915:SF325 Gene3D CH domain superfamily 1.20.58.1940 PH-like domain superfamily 1.20.58.60 CDD cd00176 cd10571 Calponin homology domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2388 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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