https://www.alphaknockout.com Mouse Brcc3 Knockout Project (CRISPR/Cas9) Objective: To create a Brcc3 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Brcc3 gene (NCBI Reference Sequence: NM_001166457 ; Ensembl: ENSMUSG00000031201 ) is located on Mouse chromosome X. 11 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000033544). Exon 5~7 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 5 starts from about 36.2% of the coding region. Exon 5~7 covers 26.69% of the coding region. The size of effective KO region: ~2527 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 5 6 7 11 Legends Exon of mouse Brcc3 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 2000 bp section upstream of Exon 5 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 2000 bp section downstream of Exon 7 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(2000bp) | A(31.45% 629) | C(17.35% 347) | T(33.75% 675) | G(17.45% 349) Note: The 2000 bp section upstream of Exon 5 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(18.3% 366) | T(35.05% 701) | G(18.9% 378) Note: The 2000 bp section downstream of Exon 7 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 2000 1 2000 2000 100.0% chrX + 75433466 75435465 2000 browser details YourSeq 155 298 1655 2000 88.3% chr11 - 78944234 79221483 277250 browser details YourSeq 93 1379 1477 2000 97.0% chr4 - 40563644 40563742 99 browser details YourSeq 92 1179 1652 2000 76.7% chr14 + 50832482 50832844 363 browser details YourSeq 83 1540 1655 2000 89.7% chr19 + 15096424 15096543 120 browser details YourSeq 82 1540 1655 2000 90.2% chr13 - 107040645 107040763 119 browser details YourSeq 80 1540 1655 2000 86.4% chr6 - 129355255 129355375 121 browser details YourSeq 79 1540 1655 2000 86.4% chr3 - 58225056 58225177 122 browser details YourSeq 79 1534 1655 2000 83.8% chr8 + 16018738 16018862 125 browser details YourSeq 78 1179 1655 2000 71.5% chr9 - 117792831 117793164 334 browser details YourSeq 78 1540 1654 2000 88.3% chr10 + 5041919 5042036 118 browser details YourSeq 77 1540 1655 2000 91.4% chr16 - 37823720 37823838 119 browser details YourSeq 77 1540 1655 2000 86.0% chr4 + 106445608 106445728 121 browser details YourSeq 77 1540 1655 2000 88.7% chr15 + 13796820 13796937 118 browser details YourSeq 76 1540 1654 2000 87.3% chrX - 14495030 14495147 118 browser details YourSeq 76 1553 1655 2000 88.8% chr11 - 56821374 56821477 104 browser details YourSeq 75 1524 1655 2000 83.2% chr14 + 68669252 68669713 462 browser details YourSeq 74 1540 1655 2000 83.7% chr8 + 45675587 45675706 120 browser details YourSeq 74 1553 1656 2000 85.6% chr2 + 100861045 100861148 104 browser details YourSeq 74 1574 1683 2000 85.5% chr17 + 4485615 4485814 200 Note: The 2000 bp section upstream of Exon 5 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% chrX + 75437993 75439992 2000 browser details YourSeq 383 60 636 2000 85.7% chr8 - 10488241 10488829 589 browser details YourSeq 378 60 640 2000 84.5% chr5 + 72628733 72629325 593 browser details YourSeq 346 57 630 2000 87.1% chr10 - 53457331 53457907 577 browser details YourSeq 345 60 538 2000 88.8% chr4 + 119398471 119398947 477 browser details YourSeq 343 60 638 2000 87.1% chr5 + 11947674 11948252 579 browser details YourSeq 342 61 589 2000 86.1% chr15 + 33503846 33504368 523 browser details YourSeq 340 60 637 2000 86.9% chr15 + 37089141 37089757 617 browser details YourSeq 338 60 637 2000 82.4% chr11 - 26660961 26661525 565 browser details YourSeq 333 57 630 2000 87.2% chr14 - 58790623 58791191 569 browser details YourSeq 332 60 630 2000 87.9% chr11 + 74710778 74711361 584 browser details YourSeq 326 57 637 2000 85.5% chr10 - 115789349 115789911 563 browser details YourSeq 321 63 637 2000 84.8% chr12 - 4218524 4219032 509 browser details YourSeq 316 60 620 2000 88.0% chr3 - 46676869 46677657 789 browser details YourSeq 313 60 526 2000 85.7% chr18 - 10896711 10897169 459 browser details YourSeq 312 60 572 2000 87.0% chr7 - 73711669 73712168 500 browser details YourSeq 303 60 526 2000 82.5% chr1 - 45363486 45363949 464 browser details YourSeq 301 65 589 2000 87.1% chr5 + 4338066 4338578 513 browser details YourSeq 298 60 567 2000 86.6% chr7 - 74963672 74964167 496 browser details YourSeq 298 52 637 2000 86.6% chr11 + 42778379 42779101 723 Note: The 2000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Brcc3 BRCA1/BRCA2-containing complex, subunit 3 [ Mus musculus (house mouse) ] Gene ID: 210766, updated on 24-Oct-2019 Gene summary Official Symbol Brcc3 provided by MGI Official Full Name BRCA1/BRCA2-containing complex, subunit 3 provided by MGI Primary source MGI:MGI:2389572 See related Ensembl:ENSMUSG00000031201 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 C6.1A Expression Broad expression in CNS E11.5 (RPKM 5.2), liver E14 (RPKM 5.1) and 24 other tissues See more Orthologs human all Genomic context Location: X; X A7.3 See Brcc3 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (75416628..75455702) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (72661967..72701040) Chromosome X - NC_000086.7 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Brcc3 ENSMUSG00000031201 Description BRCA1/BRCA2-containing complex, subunit 3 [Source:MGI Symbol;Acc:MGI:2389572] Gene Synonyms C6.1A Location Chromosome X: 75,416,628-75,454,001 forward strand. GRCm38:CM001013.2 About this gene This gene has 7 transcripts (splice variants), 211 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Brcc3-201 ENSMUST00000033544.13 4310 291aa ENSMUSP00000033544.7 Protein coding CCDS41031 P46737 TSL:1 GENCODE basic APPRIS P2 Brcc3-202 ENSMUST00000114074.7 2473 291aa ENSMUSP00000109708.1 Protein coding CCDS41031 P46737 TSL:1 GENCODE basic APPRIS P2 Brcc3-203 ENSMUST00000118428.7 2093 247aa ENSMUSP00000114057.1 Protein coding - P46737 TSL:5 GENCODE basic APPRIS ALT2 Brcc3-205 ENSMUST00000133781.7 479 158aa ENSMUSP00000123031.1 Protein coding - A3KGA8 CDS 3' incomplete TSL:5 Brcc3-207 ENSMUST00000152228.2 473 50aa ENSMUSP00000127772.1 Protein coding - E9Q0P6 CDS 3' incomplete TSL:5 Brcc3-204 ENSMUST00000124321.1 2614 No protein - Retained intron - - TSL:1 Brcc3-206 ENSMUST00000151938.7 2521 No protein - Retained intron - - TSL:2 Page 7 of 9 https://www.alphaknockout.com 57.37 kb Forward strand 75.41Mb 75.42Mb 75.43Mb 75.44Mb 75.45Mb 75.46Mb Genes Brcc3-201 >protein coding (Comprehensive set... Brcc3-203 >protein coding Gm15385-201 >unprocessed pseudogene Brcc3-206 >retained intron Gm15379-201 >processed pseudogene Brcc3-202 >protein coding Brcc3-205 >protein coding Brcc3-207 >protein coding Brcc3-204 >retained intron Contigs AL671860.6 > Genes < Cmc4-201protein coding (Comprehensive set... < Cmc4-203protein coding < Cmc4-202protein coding < Mtcp1-201protein coding < Mtcp1-203lncRNA < Mtcp1-202protein coding < Mtcp1-204retained intron Regulatory Build 75.41Mb 75.42Mb 75.43Mb 75.44Mb 75.45Mb 75.46Mb Reverse strand 57.37 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene pseudogene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000033544 37.37 kb Forward strand Brcc3-201 >protein coding ENSMUSP00000033... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF102712 SMART JAB1/MPN/MOV34 metalloenzyme domain Pfam JAB1/MPN/MOV34 metalloenzyme domain BRCC36, C-terminal helical domain PROSITE profiles MPN domain PANTHER PTHR10410 PTHR10410:SF20 Gene3D 3.40.140.10 CDD Brcc36 isopeptidase All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 240 291 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.