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Mouse Nktr Knockout Project (CRISPR/Cas9)

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Mouse Nktr Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Nktr Knockout Project (CRISPR/Cas9) Objective: To create a Nktr knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Nktr gene (NCBI Reference Sequence: NM_010918 ; Ensembl: ENSMUSG00000032525 ) is located on Mouse chromosome 9. 17 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 17 (Transcript: ENSMUST00000035112). Exon 9~15 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 9 starts from about 12.64% of the coding region. Exon 9~15 covers 83.09% of the coding region. The size of effective KO region: ~9899 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 9 10 11 12 13 14 15 17 Legends Exon of mouse Nktr 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 821 bp section upstream of Exon 9 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 527 bp section downstream of Exon 15 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(821bp) | A(20.83% 171) | C(24.48% 201) | T(34.59% 284) | G(20.1% 165) Note: The 821 bp section upstream of Exon 9 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(527bp) | A(28.65% 151) | C(19.92% 105) | T(32.64% 172) | G(18.79% 99) Note: The 527 bp section downstream of Exon 15 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 821 1 821 821 100.0% chr9 + 121741745 121742565 821 browser details YourSeq 26 785 810 821 100.0% chr3 + 7867266 7867291 26 browser details YourSeq 25 344 372 821 85.8% chr1 - 108485435 108485462 28 browser details YourSeq 25 344 372 821 85.8% chr1 - 100061170 100061197 28 browser details YourSeq 24 479 505 821 96.2% chr10 + 118309561 118309587 27 browser details YourSeq 21 206 226 821 100.0% chr5 - 129362461 129362481 21 browser details YourSeq 21 549 569 821 100.0% chr10 - 24759377 24759397 21 browser details YourSeq 21 351 377 821 88.9% chr11 + 36303117 36303143 27 browser details YourSeq 21 351 373 821 95.7% chr1 + 186648779 186648801 23 Note: The 821 bp section upstream of Exon 9 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 527 1 527 527 100.0% chr9 + 121752465 121752991 527 browser details YourSeq 26 47 76 527 93.4% chr19 - 4093665 4093694 30 browser details YourSeq 25 175 207 527 96.3% chr4 + 114851320 114851353 34 browser details YourSeq 25 49 73 527 100.0% chr11 + 70876626 70876650 25 browser details YourSeq 24 49 76 527 92.9% chr9 - 61007210 61007237 28 browser details YourSeq 24 49 76 527 92.9% chrX + 53001169 53001196 28 browser details YourSeq 24 49 74 527 96.2% chr9 + 108020516 108020541 26 browser details YourSeq 24 49 76 527 92.9% chr5 + 122363909 122363936 28 browser details YourSeq 24 52 75 527 100.0% chr2 + 61724739 61724762 24 browser details YourSeq 24 49 76 527 92.9% chr16 + 4589365 4589392 28 browser details YourSeq 24 49 76 527 92.9% chr1 + 42043750 42043777 28 browser details YourSeq 23 280 302 527 100.0% chr3 + 61453374 61453396 23 browser details YourSeq 22 51 72 527 100.0% chr3 + 41812885 41812906 22 browser details YourSeq 21 170 190 527 100.0% chr7 - 109237641 109237661 21 browser details YourSeq 21 49 69 527 100.0% chr14 - 80490871 80490891 21 browser details YourSeq 21 52 72 527 100.0% chr12 - 78886796 78886816 21 browser details YourSeq 21 239 261 527 95.7% chr14 + 53149254 53149276 23 browser details YourSeq 21 307 327 527 100.0% chr13 + 106189873 106189893 21 browser details YourSeq 20 163 184 527 95.5% chr12 - 89575595 89575616 22 browser details YourSeq 20 165 184 527 100.0% chr13 + 90955439 90955458 20 Note: The 527 bp section downstream of Exon 15 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Nktr natural killer tumor recognition sequence [ Mus musculus (house mouse) ] Gene ID: 18087, updated on 10-Oct-2019 Gene summary Official Symbol Nktr provided by MGI Official Full Name natural killer tumor recognition sequence provided by MGI Primary source MGI:MGI:97346 See related Ensembl:ENSMUSG00000032525 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 D9Wsu172e; 5330401F18Rik Expression Ubiquitous expression in limb E14.5 (RPKM 21.4), CNS E14 (RPKM 18.6) and 28 other tissues See more Orthologs human all Genomic context Location: 9 F4; 9 72.57 cM See Nktr in Genome Data Viewer Exon count: 20 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (121713881..121756842) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (121628299..121665959) Chromosome 9 - NC_000075.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 18 transcripts Gene: Nktr ENSMUSG00000032525 Description natural killer tumor recognition sequence [Source:MGI Symbol;Acc:MGI:97346] Gene Synonyms 5330401F18Rik, D9Wsu172e Location Chromosome 9: 121,719,169-121,756,843 forward strand. GRCm38:CM001002.2 About this gene This gene has 18 transcripts (splice variants), 202 orthologues, 17 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Nktr- ENSMUST00000035112.12 7022 1453aa ENSMUSP00000035112.5 Protein coding CCDS23636 P30415 TSL:1 201 GENCODE basic APPRIS P1 Nktr- ENSMUST00000182225.7 2942 966aa ENSMUSP00000138168.1 Protein coding - Q8CBP6 CDS 3' 205 incomplete TSL:1 Nktr- ENSMUST00000182713.1 811 95aa ENSMUSP00000138587.1 Protein coding - S4R2C2 CDS 5' 211 incomplete TSL:3 Nktr- ENSMUST00000182337.1 452 101aa ENSMUSP00000138164.1 Protein coding - S4R1C3 CDS 3' 207 incomplete TSL:3 Nktr- ENSMUST00000182311.7 407 99aa ENSMUSP00000138690.1 Protein coding - S4R2L3 CDS 3' 206 incomplete TSL:5 Nktr- ENSMUST00000183070.7 2576 105aa ENSMUSP00000138611.1 Nonsense mediated - Q3TQ51 TSL:1 214 decay Nktr- ENSMUST00000182179.7 1460 105aa ENSMUSP00000138437.1 Nonsense mediated - Q3TQ51 TSL:1 204 decay Nktr- ENSMUST00000182763.7 717 69aa ENSMUSP00000138272.1 Nonsense mediated - S4R1L7 TSL:5 212 decay Nktr- ENSMUST00000182503.7 539 100aa ENSMUSP00000138463.1 Nonsense mediated - S4R220 CDS 5' 209 decay incomplete TSL:3 Nktr- ENSMUST00000182904.8 8950 No - Retained intron - - TSL:1 213 protein Nktr- ENSMUST00000183311.7 2548 No - Retained intron - - TSL:2 216 protein Nktr- ENSMUST00000216573.1 1065 No - Retained intron - - TSL:2 218 protein Nktr- ENSMUST00000182607.1 789 No - Retained intron - - TSL:3 210 protein Nktr- ENSMUST00000213351.1 741 No - Retained intron - - TSL:2 217 protein Nktr- ENSMUST00000182086.1 703 No - Retained intron - - TSL:3 203 protein Nktr- ENSMUST00000182076.1 678 No - Retained intron - - TSL:2 202 protein Nktr- ENSMUST00000182395.1 417 No - Retained intron - - TSL:3 208 protein Nktr- ENSMUST00000183207.1 268 No - lncRNA - - TSL:5 215 protein Page 7 of 9 https://www.alphaknockout.com 57.67 kb Forward strand 121.71Mb 121.72Mb 121.73Mb 121.74Mb 121.75Mb 121.76Mb Genes (Comprehensive set... Ss18l2-201 >protein coding Nktr-214 >nonsense mediated decay Nktr-217 >retained intron Zfp651-204 >retained intron Ss18l2-206 >protein coding Nktr-201 >protein coding Zfp651-201 >protein coding Ss18l2-203 >retained intronNktr-212 >nonsense mediated decay Nktr-209 >nonsense mediated decay Ss18l2-205 >protein coding Nktr-213 >retained intron Nktr-218 >retained intron Nktr-210 >retained intron Ss18l2-202 >retained intronNktr-206 >protein coding Nktr-211 >protein coding Ss18l2-204 >protein coding Nktr-216 >retained intron Nktr-203 >retained intron Nktr-202 >retained intron Ss18l2-207 >retained intronNktr-215 >lncRNA Nktr-208 >retained intron Zfp651-203 >nonsense mediated decay Nktr-204 >nonsense mediated decay Nktr-205 >protein coding Nktr-207 >protein coding Contigs < AC159810.2 < AC165080.4 Genes < E530011L22Rik-201lncRNA (Comprehensive set... < E530011L22Rik-202lncRNA Regulatory Build 121.71Mb 121.72Mb 121.73Mb 121.74Mb 121.75Mb 121.76Mb Reverse strand 57.67 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site 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: ENSMUST00000035112 37.67 kb Forward strand Nktr-201 >protein coding ENSMUSP00000035..
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