https://www.alphaknockout.com

Mouse Sptbn5 Knockout Project (CRISPR/Cas9)

Objective: To create a Sptbn5 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Sptbn5 (NCBI Reference Sequence: NM_001370938 ; Ensembl: ENSMUSG00000074899 ) is located on Mouse 2. 66 exons are identified , with the ATG start codon in exon 1 and the TAA stop codon in exon 66 (Transcript: ENSMUST00000156159). 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:

Exon 3 starts from about 2.58% of the coding region. Exon 3~12 covers 20.65% of the coding region. The size of effective KO region: ~8308 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 7 8 9 10 11 12 66

Legends Exon of mouse Sptbn5 Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 1964 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 1014 bp section downstream of Exon 12 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(1964bp) | A(26.17% 514) | C(24.08% 473) | T(22.96% 451) | G(26.78% 526)

Note: The 1964 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(1014bp) | A(21.6% 219) | C(27.91% 283) | T(26.33% 267) | G(24.16% 245)

Note: The 1014 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 1964 1 1964 1964 100.0% chr2 - 120083275 120085238 1964 browser details YourSeq 38 498 660 1964 88.0% chr8 + 38790931 38791093 163 browser details YourSeq 32 495 544 1964 92.2% chr1 - 21280832 21280886 55 browser details YourSeq 29 496 534 1964 87.2% chr5 - 76362491 76362529 39 browser details YourSeq 28 499 532 1964 91.2% chr2 - 54450798 54450831 34 browser details YourSeq 28 497 534 1964 86.9% chr10 + 111571839 111571876 38 browser details YourSeq 27 495 533 1964 84.7% chr2 + 172279696 172279734 39 browser details YourSeq 25 632 659 1964 96.5% chr18 - 67748026 67748059 34 browser details YourSeq 25 498 534 1964 83.8% chr4 + 32611985 32612021 37 browser details YourSeq 24 665 690 1964 96.2% chr9 + 30907556 30907581 26 browser details YourSeq 24 498 533 1964 83.4% chr6 + 119342848 119342883 36 browser details YourSeq 23 497 531 1964 82.9% chr1 - 36679327 36679361 35 browser details YourSeq 23 499 533 1964 82.9% chr17 + 77471371 77471405 35 browser details YourSeq 23 637 659 1964 100.0% chr13 + 80918981 80919003 23 browser details YourSeq 22 618 639 1964 100.0% chr13 + 100302336 100302357 22 browser details YourSeq 21 501 533 1964 81.9% chr7 + 122550431 122550463 33 browser details YourSeq 21 618 638 1964 100.0% chr13 + 100228631 100228651 21 browser details YourSeq 21 498 534 1964 78.4% chr13 + 31589920 31589956 37

Note: The 1964 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 1014 1 1014 1014 100.0% chr2 - 120073953 120074966 1014 browser details YourSeq 31 482 529 1014 82.9% chr11 + 34065276 34065320 45 browser details YourSeq 31 194 251 1014 97.1% chr10 + 71443479 71443907 429 browser details YourSeq 26 133 160 1014 100.0% chr1 - 108731480 108731521 42 browser details YourSeq 23 514 539 1014 96.0% chr10 - 85036071 85036106 36 browser details YourSeq 22 95 117 1014 100.0% chr1 + 17590720 17590743 24

Note: The 1014 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Sptbn5 beta, non-erythrocytic 5 [ Mus musculus (house mouse) ] Gene ID: 640524, updated on 10-Oct-2019

Gene summary

Official Symbol Sptbn5 provided by MGI Official Full Name spectrin beta, non-erythrocytic 5 provided by MGI Primary source MGI:MGI:2685200 See related Ensembl:ENSMUSG00000074899 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 Gm354; Spnb5; EG640524 Expression Low expression observed in reference dataset See more Orthologs human all

Genomic context

Location: 2; 2 E5 See Sptbn5 in Genome Data Viewer Exon count: 68

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (120041493..120088913, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (119867830..119911414, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 1 transcript

Gene: Sptbn5 ENSMUSG00000074899

Description spectrin beta, non-erythrocytic 5 [Source:MGI Symbol;Acc:MGI:2685200] Gene Synonyms EG640524, Spnb5 Location Chromosome 2: 120,041,493-120,085,678 reverse strand. GRCm38:CM000995.2 About this gene This gene has 1 transcript (splice variant), 140 orthologues, 36 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS Flags

Sptbn5-201 ENSMUST00000156159.3 11654 3624aa ENSMUSP00000158705.1 Protein coding - TSL:5 GENCODE basic APPRIS P1

64.19 kb Forward strand 120.04Mb 120.05Mb 120.06Mb 120.07Mb 120.08Mb 120.09Mb Gm28042-204 >nonsense mediated decay (Comprehensive set...

Gm28042-202 >protein coding

Gm28042-203 >protein coding

Jmjd7-201 >protein codingPla2g4b-202 >retained intron

Jmjd7-203 >processed transcript

Gm28042-201 >nonsense mediated decay

Pla2g4b-203 >retained intron

Pla2g4b-205 >retained intron

Pla2g4b-201 >protein coding

Pla2g4b-204 >retained intron

Pla2g4b-206 >retained intron

Contigs < AL833774.4

Genes < Sptbn5-201protein coding < Ehd4-201protein coding (Comprehensive set...

Regulatory Build

120.04Mb 120.05Mb 120.06Mb 120.07Mb 120.08Mb 120.09Mb Reverse strand 64.19 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000156159

< Sptbn5-201protein coding

Reverse strand 44.19 kb

ENSMUSP00000158... Low complexity (Seg)

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

Variant Legend

inframe deletion missense variant synonymous variant

Scale bar 0 400 800 1200 1600 2000 2400 2800 3200 3624

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

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