https://www.alphaknockout.com Mouse Nrn1 Knockout Project (CRISPR/Cas9) Objective: To create a Nrn1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Nrn1 gene (NCBI Reference Sequence: NM_153529 ; Ensembl: ENSMUSG00000039114 ) is located on Mouse chromosome 13. 3 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000037623). 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 knock-out allele exhibit a reduction in body length and body weight, delayed axonal, dendritic, and synaptic development, reduced dendritic spine maintenance leading to gradual spine loss, and impaired associative and spatial learning. Exon 2 starts from about 13.15% of the coding region. Exon 2 covers 34.04% of the coding region. The size of effective KO region: ~145 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 3 Legends Exon of mouse Nrn1 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 2000 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 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(25.95% 519) | C(25.65% 513) | T(23.8% 476) | G(24.6% 492) Note: The 2000 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(27.65% 553) | C(24.45% 489) | T(24.75% 495) | G(23.15% 463) Note: The 2000 bp section downstream 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 2000 1 2000 2000 100.0% chr13 - 36730252 36732251 2000 browser details YourSeq 75 1008 1217 2000 95.2% chr12 + 5096667 5097192 526 browser details YourSeq 50 1152 1218 2000 82.4% chr1 + 34654525 34654575 51 browser details YourSeq 47 1149 1222 2000 76.0% chr1 - 56926477 56926526 50 browser details YourSeq 24 1195 1219 2000 100.0% chrX - 170737323 170737365 43 browser details YourSeq 24 1556 1579 2000 100.0% chr11 - 20457739 20457762 24 browser details YourSeq 23 600 625 2000 96.2% chr1 - 15973880 15973906 27 browser details YourSeq 22 981 1002 2000 100.0% chr15 - 102724111 102724132 22 browser details YourSeq 22 74 95 2000 100.0% chr12 - 97350015 97350036 22 browser details YourSeq 22 977 998 2000 100.0% chr1 - 69938711 69938732 22 browser details YourSeq 21 987 1007 2000 100.0% chr13 - 58629531 58629551 21 browser details YourSeq 21 1123 1145 2000 95.7% chr1 - 49884344 49884366 23 browser details YourSeq 20 1746 1765 2000 100.0% chr1 - 22133759 22133778 20 Note: The 2000 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% chr13 - 36728107 36730106 2000 browser details YourSeq 37 1748 1828 2000 97.5% chr6 - 103555055 103555398 344 browser details YourSeq 34 1789 1823 2000 100.0% chr13 + 52881116 52881153 38 browser details YourSeq 33 921 955 2000 100.0% chr10 + 43453910 43453948 39 browser details YourSeq 29 1793 1827 2000 90.7% chr5 - 144290381 144290414 34 browser details YourSeq 27 1798 1826 2000 100.0% chr2 + 52832411 52832441 31 browser details YourSeq 24 1237 1261 2000 100.0% chr16 - 50842640 50842672 33 browser details YourSeq 22 103 125 2000 100.0% chr5 - 106615845 106615869 25 browser details YourSeq 22 1790 1813 2000 87.0% chr1 - 13133490 13133512 23 browser details YourSeq 22 96 117 2000 100.0% chr6 + 142506800 142506821 22 browser details YourSeq 22 1240 1261 2000 100.0% chr3 + 19910650 19910671 22 Note: The 2000 bp section downstream 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: Nrn1 neuritin 1 [ Mus musculus (house mouse) ] Gene ID: 68404, updated on 24-Oct-2019 Gene summary Official Symbol Nrn1 provided by MGI Official Full Name neuritin 1 provided by MGI Primary source MGI:MGI:1915654 See related Ensembl:ENSMUSG00000039114 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 Nrn; Cpg15; 0710008J23Rik Expression Biased expression in liver E18 (RPKM 84.3), cortex adult (RPKM 52.3) and 11 other tissues See more Orthologs human all Genomic context Location: 13; 13 A3.3 See Nrn1 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (36725622..36734477, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (36817494..36826323, complement) Chromosome 13 - NC_000079.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Nrn1 ENSMUSG00000039114 Description neuritin 1 [Source:MGI Symbol;Acc:MGI:1915654] Gene Synonyms 0710008J23Rik, cpg15 Location Chromosome 13: 36,725,361-36,735,131 reverse strand. GRCm38:CM001006.2 About this gene This gene has 5 transcripts (splice variants), 193 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 13 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Nrn1-201 ENSMUST00000037623.14 1619 142aa ENSMUSP00000040900.8 Protein coding CCDS26455 Q8CFV4 TSL:1 GENCODE basic APPRIS P1 Nrn1-204 ENSMUST00000224323.1 1779 151aa ENSMUSP00000153099.1 Protein coding - A0A286YD56 GENCODE basic Nrn1-203 ENSMUST00000223611.1 1443 94aa ENSMUSP00000153395.1 Protein coding - A0A286YDV9 GENCODE basic Nrn1-202 ENSMUST00000122286.1 691 158aa ENSMUSP00000113721.1 Protein coding - D3Z4E2 TSL:3 GENCODE basic Nrn1-205 ENSMUST00000224960.1 559 140aa ENSMUSP00000153173.1 Protein coding - A0A286YCW7 CDS 3' incomplete 29.77 kb Forward strand 36.72Mb 36.73Mb 36.74Mb Genes Fars2-210 >nonsense mediated decay Gm30177-201 >lncRNA (Comprehensive set... Fars2-211 >lncRNA Contigs < AC156802.7 Genes (Comprehensive set... < Nrn1-204protein coding < Nrn1-201protein coding < Nrn1-203protein coding < Nrn1-202protein coding < Nrn1-205protein coding Regulatory Build 36.72Mb 36.73Mb 36.74Mb Reverse strand 29.77 kb Regulation Legend Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000037623 < Nrn1-201protein coding Reverse strand 8.85 kb ENSMUSP00000040... Low complexity (Seg) Cleavage site (Sign... Pfam Neuritin family PANTHER Neuritin family PTHR15902:SF1 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 20 40 60 80 100 120 142 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.