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Mouse Rtn1 Knockout Project (CRISPR/Cas9)

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Mouse Rtn1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rtn1 Knockout Project (CRISPR/Cas9) Objective: To create a Rtn1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rtn1 gene (NCBI Reference Sequence: NM_153457 ; Ensembl: ENSMUSG00000021087 ) is located on Mouse chromosome 12. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000078505). Exon 2 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 are viable with no gross abnormalities. Exon 2 starts from about 10.34% of the coding region. Exon 2 covers 33.08% of the coding region. The size of effective KO region: ~774 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 9 Legends Exon of mouse Rtn1 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 774 bp section of Exon 2 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 774 bp section of Exon 2 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 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(774bp) | A(28.04% 217) | C(28.04% 217) | T(19.9% 154) | G(24.03% 186) Note: The 774 bp section of Exon 2 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(774bp) | A(28.04% 217) | C(28.04% 217) | T(19.9% 154) | G(24.03% 186) Note: The 774 bp section of Exon 2 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 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 774 1 774 774 100.0% chr12 - 72308155 72308928 774 browser details YourSeq 23 233 258 774 83.4% chr11 + 80322768 80322791 24 browser details YourSeq 22 391 415 774 95.9% chr11 - 95381217 95381242 26 browser details YourSeq 21 715 735 774 100.0% chr12 - 87766138 87766158 21 browser details YourSeq 21 715 735 774 100.0% chr1 - 95256657 95256677 21 browser details YourSeq 21 715 735 774 100.0% chr12 + 88089267 88089287 21 browser details YourSeq 20 352 371 774 100.0% chr14 + 123196271 123196290 20 Note: The 774 bp section of Exon 2 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 774 1 774 774 100.0% chr12 - 72308157 72308930 774 browser details YourSeq 22 393 417 774 95.9% chr11 - 95381217 95381242 26 browser details YourSeq 21 717 737 774 100.0% chr12 - 87766138 87766158 21 browser details YourSeq 21 717 737 774 100.0% chr1 - 95256657 95256677 21 browser details YourSeq 21 717 737 774 100.0% chr12 + 88089267 88089287 21 browser details YourSeq 20 354 373 774 100.0% chr14 + 123196271 123196290 20 Note: The 774 bp section of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Rtn1 reticulon 1 [ Mus musculus (house mouse) ] Gene ID: 104001, updated on 12-Aug-2019 Gene summary Official Symbol Rtn1 provided by MGI Official Full Name reticulon 1 provided by MGI Primary source MGI:MGI:1933947 See related Ensembl:ENSMUSG00000021087 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 Nsp Expression Biased expression in CNS E18 (RPKM 186.8), whole brain E14.5 (RPKM 137.8) and 7 other tissues See more Orthologs human all Genomic context Location: 12; 12 C3 See Rtn1 in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (72211749..72409054, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (73312739..73509926, complement) Chromosome 12 - NC_000078.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Rtn1 ENSMUSG00000021087 Description reticulon 1 [Source:MGI Symbol;Acc:MGI:1933947] Gene Synonyms 0710005K15Rik, 4930441F12Rik, Nsp, Rtn1-a, Rtn1-b, Rtn1-c Location Chromosome 12: 72,211,752-72,409,054 reverse strand. GRCm38:CM001005.2 About this gene This gene has 6 transcripts (splice variants), 209 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rtn1-202 ENSMUST00000078505.13 3629 780aa ENSMUSP00000077594.7 Protein coding CCDS25968 Q4FJL2 Q8K0T0 TSL:1 GENCODE basic Rtn1-201 ENSMUST00000021497.15 1544 208aa ENSMUSP00000021497.9 Protein coding CCDS25969 Q7M6W1 TSL:1 GENCODE basic APPRIS P1 Rtn1-203 ENSMUST00000137990.1 1008 253aa ENSMUSP00000120033.1 Protein coding - A3QM89 CDS 5' incomplete TSL:1 Rtn1-206 ENSMUST00000221756.1 1076 No protein - Retained intron - - TSL:NA Rtn1-205 ENSMUST00000150156.1 1428 No protein - lncRNA - - TSL:1 Rtn1-204 ENSMUST00000144181.1 465 No protein - lncRNA - - TSL:2 217.30 kb Forward strand 72.25Mb 72.30Mb 72.35Mb 72.40Mb Contigs CT010476.10 > AC103353.7 > Genes (Comprehensive set... < Rtn1-202protein coding < Rtn1-205lncRNA < Rtn1-201protein coding < Rtn1-203protein coding < Rtn1-204lncRNA < Rtn1-206retained intron Regulatory Build 72.25Mb 72.30Mb 72.35Mb 72.40Mb Reverse strand 217.30 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 RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000078505 < Rtn1-202protein coding Reverse strand 197.30 kb ENSMUSP00000077... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam Reticulon PROSITE profiles Reticulon PANTHER PTHR45799:SF5 PTHR45799 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 780 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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