https://www.alphaknockout.com Mouse Nars Knockout Project (CRISPR/Cas9) Objective: To create a Nars knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Nars gene (NCBI Reference Sequence: NM_001142950 ; Ensembl: ENSMUSG00000024587 ) is located on Mouse chromosome 18. 15 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 15 (Transcript: ENSMUST00000237400). Exon 2~8 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 2 starts from about 2.62% of the coding region. Exon 2~8 covers 33.93% of the coding region. The size of effective KO region: ~7637 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 2 3 4 5 6 7 8 15 Legends Exon of mouse Nars 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 1007 bp section upstream of Exon 2 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 8 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(1007bp) | A(25.22% 254) | C(20.75% 209) | T(28.9% 291) | G(25.12% 253) Note: The 1007 bp section upstream 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(2000bp) | A(27.75% 555) | C(18.95% 379) | T(31.05% 621) | G(22.25% 445) Note: The 2000 bp section downstream of Exon 8 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 1007 1 1007 1007 100.0% chr18 - 64515409 64516415 1007 browser details YourSeq 29 392 439 1007 94.0% chr11 - 74846620 74846885 266 browser details YourSeq 27 755 786 1007 96.6% chr1 - 128728091 128728130 40 browser details YourSeq 23 765 787 1007 100.0% chr10 - 115018413 115018435 23 browser details YourSeq 23 757 779 1007 100.0% chr7 + 53927374 53927396 23 browser details YourSeq 22 348 369 1007 100.0% chr11 + 40632225 40632246 22 browser details YourSeq 21 546 566 1007 100.0% chr12 + 99378696 99378716 21 browser details YourSeq 21 391 411 1007 100.0% chr1 + 187931108 187931128 21 browser details YourSeq 20 980 999 1007 100.0% chr12 - 8342943 8342962 20 browser details YourSeq 20 845 864 1007 100.0% chr12 + 4836106 4836125 20 Note: The 1007 bp section upstream 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 2000 1 2000 2000 100.0% chr18 - 64505772 64507771 2000 browser details YourSeq 448 446 1988 2000 91.2% chr16 - 31076995 31579827 502833 browser details YourSeq 398 427 1989 2000 92.5% chr15 + 76153768 76316869 163102 browser details YourSeq 230 448 772 2000 90.7% chr5 - 147622346 147622689 344 browser details YourSeq 210 445 761 2000 87.9% chr9 + 59189816 59190159 344 browser details YourSeq 205 447 772 2000 87.3% chr7 - 99294419 99294750 332 browser details YourSeq 205 427 765 2000 86.8% chr2 - 92465010 92465343 334 browser details YourSeq 202 443 772 2000 89.7% chr3 + 53094891 53095268 378 browser details YourSeq 202 433 756 2000 88.5% chr18 + 65773165 65773502 338 browser details YourSeq 201 408 756 2000 88.4% chr5 + 116184431 116184777 347 browser details YourSeq 199 427 715 2000 90.5% chr11 + 109521969 109522260 292 browser details YourSeq 198 427 755 2000 86.7% chr7 + 39627505 39627848 344 browser details YourSeq 197 427 767 2000 89.6% chrX + 7208874 7209234 361 browser details YourSeq 196 427 716 2000 87.8% chr17 - 75481971 75482285 315 browser details YourSeq 195 407 714 2000 89.3% chr6 - 137861455 137861802 348 browser details YourSeq 195 427 772 2000 88.7% chr15 + 79241576 79241961 386 browser details YourSeq 192 422 732 2000 89.7% chr2 - 120886547 120886871 325 browser details YourSeq 191 415 723 2000 90.1% chr2 + 118338645 118338972 328 browser details YourSeq 191 443 718 2000 89.9% chr10 + 83762900 83763168 269 browser details YourSeq 190 443 772 2000 92.2% chr12 - 55596233 55596582 350 Note: The 2000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Nars asparaginyl-tRNA synthetase [ Mus musculus (house mouse) ] Gene ID: 70223, updated on 24-Oct-2019 Gene summary Official Symbol Nars provided by MGI Official Full Name asparaginyl-tRNA synthetase provided by MGI Primary source MGI:MGI:1917473 See related Ensembl:ENSMUSG00000024587 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 ASNRS; C78150; AA960128; 3010001M15Rik Expression Ubiquitous expression in liver E14 (RPKM 51.7), placenta adult (RPKM 44.5) and 28 other tissues See more Orthologs human all Genomic context Location: 18; 18 E1 See Nars in Genome Data Viewer Exon count: 15 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (64499647..64516557, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (64659301..64676211, complement) Chromosome 18 - NC_000084.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 16 transcripts Gene: Nars ENSMUSG00000024587 Description asparaginyl-tRNA synthetase [Source:MGI Symbol;Acc:MGI:1917473] Gene Synonyms ASNRS Location Chromosome 18: 64,499,647-64,516,652 reverse strand. GRCm38:CM001011.2 About this gene This gene has 16 transcripts (splice variants), 236 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Nars- ENSMUST00000237400.1 2745 559aa ENSMUSP00000157402.1 Protein coding CCDS50308 Q8BP47 GENCODE 213 basic APPRIS P2 Nars- ENSMUST00000025483.10 2687 558aa ENSMUSP00000025483.10 Protein coding - - TSL:1 201 GENCODE basic APPRIS ALT1 Nars- ENSMUST00000236186.1 1784 517aa ENSMUSP00000158356.1 Protein coding - A0A494BB89 GENCODE 205 basic Nars- ENSMUST00000237351.1 1492 497aa ENSMUSP00000158278.1 Protein coding - A0A494BAX5 CDS 5' 211 incomplete Nars- ENSMUST00000235325.1 761 210aa ENSMUSP00000157468.1 Protein coding - A0A494B927 CDS 3' 202 incomplete Nars- ENSMUST00000236873.1 2482 127aa ENSMUSP00000157502.1 Nonsense mediated - A0A494B996 - 209 decay Nars- ENSMUST00000237369.1 2103 70aa ENSMUSP00000157488.1 Nonsense mediated - A0A494B949 - 212 decay Nars- ENSMUST00000236392.1 935 164aa ENSMUSP00000158211.1 Nonsense mediated - A0A494BAW6 - 206 decay Nars- ENSMUST00000236463.1 894 43aa ENSMUSP00000158367.1 Nonsense mediated - A0A494BB92 - 207 decay Nars- ENSMUST00000235647.1 874 69aa ENSMUSP00000157626.1 Nonsense mediated - A0A494B9G0 - 203 decay Nars- ENSMUST00000236583.1 845 35aa ENSMUSP00000158408.1 Nonsense mediated - A0A494BB81 - 208 decay Nars- ENSMUST00000237585.1 2268 No - Retained intron - - - 215 protein Nars- ENSMUST00000237503.1 1648 No - Retained intron - - - 214 protein Nars- ENSMUST00000237027.1 848 No - Retained intron - - - 210 protein Nars- ENSMUST00000237831.1 774 No - Retained intron - - - 216 protein Nars- ENSMUST00000235887.1 434 No - Retained intron - - - 204 protein Page 7 of 9 https://www.alphaknockout.com 37.01 kb Forward strand 64.49Mb 64.50Mb 64.51Mb 64.52Mb Contigs AC102268.11 > Genes (Comprehensive set... < Fech-209protein coding < Nars-213protein coding < Nars-209nonsense mediated decay < Nars-214retained intron< Nars-206nonsense mediated decay < Nars-201protein coding < Nars-212nonsense mediated decay < Nars-215retained intron < Nars-211protein coding < Nars-205protein coding < Nars-204retained intron < Nars-210retained intron < Nars-216retained intron < Nars-208nonsense mediated decay < Nars-203nonsense mediated decay < Nars-207nonsense mediated decay < Nars-202protein coding Regulatory Build 64.49Mb 64.50Mb 64.51Mb 64.52Mb Reverse strand 37.01 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 processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000237400 < Nars-213protein