https://www.alphaknockout.com Mouse Acrv1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Acrv1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Acrv1 gene (NCBI Reference Sequence: NM_007391 ; Ensembl: ENSMUSG00000032110 ) is located on Mouse chromosome 9. 4 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 4 (Transcript: ENSMUST00000034620). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Acrv1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-339F21 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 2~3 is not frameshift exon, and covers 78.16% of the coding region. The size of intron 1 for 5'-loxP site insertion: 847 bp, and the size of intron 3 for 3'-loxP site insertion: 1789 bp. The size of effective cKO region: ~2999 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Acrv1 cKO region loxP site Page 2 of 7 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(9499bp) | A(32.42% 3080) | C(20.33% 1931) | T(27.15% 2579) | G(20.1% 1909) Note: The sequence of homologous arms and cKO region 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 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr9 + 36690959 36693958 3000 browser details YourSeq 70 12 119 3000 98.7% chr9 - 93193868 93194144 277 browser details YourSeq 25 223 257 3000 88.9% chr4 - 144884216 144884249 34 Note: The 3000 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 3000 1 3000 3000 100.0% chr9 + 36696958 36699957 3000 browser details YourSeq 333 2262 2860 3000 89.3% chr7 - 141914442 141915112 671 browser details YourSeq 281 2212 2999 3000 86.3% chr19 + 11825544 11826336 793 browser details YourSeq 209 2170 2548 3000 88.9% chr1 + 176172418 176172871 454 browser details YourSeq 205 2209 2548 3000 90.2% chr1 - 170066560 170066962 403 browser details YourSeq 203 2171 2541 3000 90.0% chr9 - 83525781 83526173 393 browser details YourSeq 202 2693 3000 3000 87.2% chr19 - 17569036 17569348 313 browser details YourSeq 202 2220 2927 3000 84.0% chr18 - 21515529 21516246 718 browser details YourSeq 197 2166 2548 3000 85.7% chr6 + 148837554 148837971 418 browser details YourSeq 195 2225 2548 3000 89.7% chr1 - 55200068 55602057 401990 browser details YourSeq 190 2315 2900 3000 86.8% chr4 - 97470257 97470868 612 browser details YourSeq 188 2181 2544 3000 89.8% chr17 + 83777734 83778129 396 browser details YourSeq 187 2192 2561 3000 89.1% chr1 - 51669111 51669517 407 browser details YourSeq 181 2502 2865 3000 86.4% chr17 + 93152342 93152773 432 browser details YourSeq 179 2533 2987 3000 79.9% chr16 + 20842935 20843426 492 browser details YourSeq 174 2171 2992 3000 90.3% chr2 + 138685718 138686556 839 browser details YourSeq 173 2653 2994 3000 89.5% chr2 + 181605308 181605860 553 browser details YourSeq 171 2177 2548 3000 89.9% chr10 - 86086434 86086912 479 browser details YourSeq 168 2541 2824 3000 85.6% chr18 - 19430202 19430485 284 browser details YourSeq 168 2255 2548 3000 90.5% chr2 + 72999315 72999812 498 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Acrv1 acrosomal vesicle protein 1 [ Mus musculus (house mouse) ] Gene ID: 11451, updated on 12-Aug-2019 Gene summary Official Symbol Acrv1 provided by MGI Official Full Name acrosomal vesicle protein 1 provided by MGI Primary source MGI:MGI:104590 See related Ensembl:ENSMUSG00000032110 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 Msa63; SP-10 Expression Restricted expression toward testis adult (RPKM 178.7) See more Orthologs human all Genomic context Location: 9 A4; 9 20.67 cM See Acrv1 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (36693220..36698845) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (36500805..36506422) Chromosome 9 - NC_000075.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Acrv1 ENSMUSG00000032110 Description acrosomal vesicle protein 1 [Source:MGI Symbol;Acc:MGI:104590] Gene Synonyms Msa63, SP-10 Location Chromosome 9: 36,693,220-36,698,843 forward strand. GRCm38:CM001002.2 About this gene This gene has 2 transcripts (splice variants), 87 orthologues, 12 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Acrv1-201 ENSMUST00000034620.4 1101 261aa ENSMUSP00000034620.3 Protein coding CCDS22971 P50289 TSL:1 GENCODE basic APPRIS P1 Acrv1-202 ENSMUST00000184160.1 3265 No protein - Retained intron - - TSL:1 25.62 kb Forward strand 36.685Mb 36.690Mb 36.695Mb 36.700Mb 36.705Mb Genes (Comprehensive set... Acrv1-201 >protein coding Acrv1-202 >retained intron Contigs < AC155921.2 Genes < Gm49367-201lncRNA < Chek1-201protein coding (Comprehensive set... < 1700027I24Rik-201lncRNA < Chek1-202protein coding < 1700027I24Rik-202lncRNA Regulatory Build 36.685Mb 36.690Mb 36.695Mb 36.700Mb 36.705Mb Reverse strand 25.62 kb Regulation Legend CTCF Enhancer Open Chromatin Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000034620 5.62 kb Forward strand Acrv1-201 >protein coding ENSMUSP00000034... MobiDB lite Low complexity (Seg) Cleavage site (Sign... CDD cd00117 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 261 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.