https://www.alphaknockout.com
Mouse Neto1 Knockout Project (CRISPR/Cas9)
Objective: To create a Neto1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Neto1 gene (NCBI Reference Sequence: NM_144946 ; Ensembl: ENSMUSG00000050321 ) is located on Mouse chromosome 18. 11 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000058829). Exon 5~6 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: Mice homozygous for a null allele exhibit depressed long term potentiation, reduced NMDAR excitatory postsynaptic potentiation, and decreased spartial learning and working memory.
Exon 5 starts from about 29.39% of the coding region. Exon 5~6 covers 10.63% of the coding region. The size of effective KO region: ~286 bp. The KO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele gRNA region 5' gRNA region 3'
1 5 6 11
Legends Exon of mouse Neto1 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 5 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 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(2000bp) | A(36.1% 722) | C(17.35% 347) | T(30.5% 610) | G(16.05% 321)
Note: The 2000 bp section upstream of Exon 5 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(32.15% 643) | C(18.6% 372) | T(27.65% 553) | G(21.6% 432)
Note: The 2000 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 2000 1 2000 2000 100.0% chr18 + 86459043 86461042 2000 browser details YourSeq 41 899 943 2000 97.8% chr11 + 5352440 5352499 60 browser details YourSeq 39 895 938 2000 88.1% chr11 + 12434981 12435022 42 browser details YourSeq 37 909 955 2000 93.1% chr8 + 31866019 31866247 229 browser details YourSeq 35 892 939 2000 92.9% chr3 - 44582544 44582606 63 browser details YourSeq 29 894 928 2000 91.5% chr11 + 56349980 56350014 35 browser details YourSeq 28 919 946 2000 100.0% chr11 - 121333424 121333451 28 browser details YourSeq 26 915 940 2000 100.0% chr2 + 161618473 161618498 26 browser details YourSeq 25 928 953 2000 100.0% chr15 - 22720411 22720457 47 browser details YourSeq 23 923 947 2000 96.0% chr2 + 90935692 90935716 25 browser details YourSeq 20 729 754 2000 88.5% chr6 - 13762371 13762396 26
Note: The 2000 bp section upstream of Exon 5 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% chr18 + 86461329 86463328 2000 browser details YourSeq 29 1247 1322 2000 76.6% chr6 - 144615672 144615746 75 browser details YourSeq 27 1305 1331 2000 100.0% chr4 - 130655960 130655986 27 browser details YourSeq 27 1266 1326 2000 87.1% chrX + 124279125 124279184 60 browser details YourSeq 26 291 332 2000 82.5% chr5 - 134073295 134073338 44 browser details YourSeq 26 1306 1334 2000 96.6% chrX + 16655031 16655068 38 browser details YourSeq 26 1301 1326 2000 100.0% chr7 + 65509021 65509046 26 browser details YourSeq 25 1342 1382 2000 80.5% chr2 + 13607673 13607713 41 browser details YourSeq 24 1301 1326 2000 96.2% chr16 + 72929661 72929686 26 browser details YourSeq 24 1306 1339 2000 85.3% chr11 + 101746513 101746546 34 browser details YourSeq 23 1301 1326 2000 96.0% chr9 - 77950715 77950741 27
Note: The 2000 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: Neto1 neuropilin (NRP) and tolloid (TLL)-like 1 [ Mus musculus (house mouse) ] Gene ID: 246317, updated on 15-Aug-2019
Gene summary
Official Symbol Neto1 provided by MGI Official Full Name neuropilin (NRP) and tolloid (TLL)-like 1 provided by MGI Primary source MGI:MGI:2180216 See related Ensembl:ENSMUSG00000050321 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 Btcl1; AI851453; C130005O10Rik Expression Biased expression in frontal lobe adult (RPKM 3.4), cortex adult (RPKM 2.9) and 4 other tissues See more Orthologs human all
Genomic context
Location: 18; 18 E4 See Neto1 in Genome Data Viewer Exon count: 14
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (86394815..86506721)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (86564344..86671289)
Chromosome 18 - NC_000084.6
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Transcript information: This gene has 3 transcripts
Gene: Neto1 ENSMUSG00000050321
Description neuropilin (NRP) and tolloid (TLL)-like 1 [Source:MGI Symbol;Acc:MGI:2180216] Gene Synonyms C130005O10Rik Location Chromosome 18: 86,394,952-86,506,718 forward strand. GRCm38:CM001011.2 About this gene This gene has 3 transcripts (splice variants), 248 orthologues, 35 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Neto1-201 ENSMUST00000058829.3 8352 533aa ENSMUSP00000057340.2 Protein coding CCDS29387 Q8R4I7 TSL:1 GENCODE basic APPRIS P1
Neto1-202 ENSMUST00000235167.1 527 68aa ENSMUSP00000158106.1 Protein coding - A0A494BAI8 CDS 5' incomplete
Neto1-203 ENSMUST00000236600.1 384 No protein - lncRNA - - -
131.77 kb Forward strand
86.40Mb 86.45Mb 86.50Mb Genes (Comprehensive set... Neto1-201 >protein coding
Neto1-203 >lncRNA Neto1-202 >protein coding
Contigs AC113599.10 > Genes < Gm50384-201lncRNA (Comprehensive set...
Regulatory Build
86.40Mb 86.45Mb 86.50Mb Reverse strand 131.77 kb
Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site
Gene Legend Protein Coding
Ensembl protein coding merged Ensembl/Havana
Non-Protein Coding
RNA gene
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Transcript: ENSMUST00000058829
111.77 kb Forward strand
Neto1-201 >protein coding
ENSMUSP00000057... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily Spermadhesin, CUB domain superfamily
LDL receptor-like superfamily SMART CUB domain Low-density lipoprotein (LDL) receptor class A repeat Pfam CUB domain Low-density lipoprotein (LDL) receptor class A repeat
PROSITE profiles CUB domain Low-density lipoprotein (LDL) receptor class A repeat PROSITE patterns Low-density lipoprotein (LDL) receptor class A, conserved site PANTHER Neuropilin and tolloid-like protein 1
PTHR45645 Gene3D Spermadhesin, CUB domain superfamily 2.40.128.620 CDD CUB domain Low-density lipoprotein (LDL) receptor class A repeat
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend
missense variant splice region variant synonymous variant
Scale bar 0 60 120 180 240 300 360 420 533
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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