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Mouse Clec3b Knockout Project (CRISPR/Cas9)

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Mouse Clec3b Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Clec3b Knockout Project (CRISPR/Cas9) Objective: To create a Clec3b knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Clec3b gene (NCBI Reference Sequence: NM_011606 ; Ensembl: ENSMUSG00000025784 ) is located on Mouse chromosome 9. 3 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 3 (Transcript: ENSMUST00000026890). Exon 1~3 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 null allele develop pronounced cervical lordosis and thoracic kyphosis associated with wedge-shaped deformities of the vertebrae, growth plate irregularities, and an asymmetric development of the intervertebral disks. Exon 1 starts from about 0.17% of the coding region. Exon 1~3 covers 100.0% of the coding region. The size of effective KO region: ~6101 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 Clec3b 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 start codon 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 stop codon 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(22.85% 457) | C(24.85% 497) | T(27.95% 559) | G(24.35% 487) Note: The 2000 bp section upstream of start codon 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(23.95% 479) | C(24.65% 493) | T(26.7% 534) | G(24.7% 494) Note: The 2000 bp section downstream of stop codon 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% chr9 + 123149045 123151044 2000 browser details YourSeq 435 551 1029 2000 95.6% chr8 - 14085456 14086000 545 browser details YourSeq 432 552 1032 2000 95.2% chr7 - 141768675 141769215 541 browser details YourSeq 426 552 1029 2000 95.2% chr5 - 49587862 49588399 538 browser details YourSeq 425 552 1029 2000 95.4% chr11 + 58769011 58769505 495 browser details YourSeq 422 552 1029 2000 94.8% chr9 - 18910203 19093330 183128 browser details YourSeq 422 552 1030 2000 94.4% chr16 + 53484551 53485097 547 browser details YourSeq 414 552 1029 2000 93.5% chr8 + 74595236 74595722 487 browser details YourSeq 414 558 1030 2000 94.3% chr5 + 10337788 10338321 534 browser details YourSeq 414 552 1031 2000 93.9% chr10 + 10653963 10654512 550 browser details YourSeq 413 552 1029 2000 93.7% chr5 + 54989461 54990001 541 browser details YourSeq 412 552 1029 2000 93.5% chr18 - 14446637 14447122 486 browser details YourSeq 411 552 1029 2000 93.5% chr16 + 5358307 5358788 482 browser details YourSeq 410 552 1030 2000 94.4% chr11 - 23787502 23788040 539 browser details YourSeq 410 552 1029 2000 96.0% chr18 + 86225413 86225899 487 browser details YourSeq 409 552 1030 2000 93.1% chr15 + 11251848 11252385 538 browser details YourSeq 408 551 1029 2000 94.6% chr10 - 26148818 26149308 491 browser details YourSeq 408 552 1050 2000 91.2% chr7 + 142764012 142764499 488 browser details YourSeq 407 552 1030 2000 92.7% chr15 + 22303266 22303741 476 browser details YourSeq 406 560 1031 2000 93.6% chr18 - 72616383 72628412 12030 Note: The 2000 bp section upstream of start codon 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% chr9 + 123157146 123159145 2000 browser details YourSeq 204 293 499 2000 99.6% chrX + 103974958 103975165 208 browser details YourSeq 202 291 498 2000 99.1% chr11 + 30656609 30656840 232 browser details YourSeq 201 291 495 2000 99.1% chr3 + 87881981 87882185 205 browser details YourSeq 200 289 502 2000 97.2% chr8 - 105914031 105914414 384 browser details YourSeq 200 292 494 2000 99.6% chr13 + 91145778 91145981 204 browser details YourSeq 200 232 484 2000 99.1% chr12 + 111142201 111142741 541 browser details YourSeq 199 292 497 2000 98.6% chr8 - 41308411 41308628 218 browser details YourSeq 199 293 510 2000 95.2% chr8 + 50187665 50187871 207 browser details YourSeq 198 292 501 2000 98.1% chr8 - 72444268 72444477 210 browser details YourSeq 198 286 498 2000 97.1% chr3 - 58585490 58585699 210 browser details YourSeq 198 291 498 2000 97.6% chr17 + 8379695 8379902 208 browser details YourSeq 197 290 490 2000 99.1% chr14 - 55696274 55696474 201 browser details YourSeq 196 290 490 2000 99.1% chr17 + 12694879 12695081 203 browser details YourSeq 195 293 499 2000 97.6% chr5 - 77268995 77269417 423 browser details YourSeq 195 292 495 2000 97.0% chr17 - 80492176 80492376 201 browser details YourSeq 195 292 500 2000 95.6% chr9 + 108659886 108660087 202 browser details YourSeq 194 292 491 2000 98.5% chr2 - 180003857 180004056 200 browser details YourSeq 194 289 500 2000 95.5% chr16 + 44390269 44390465 197 browser details YourSeq 193 290 485 2000 99.5% chr7 - 138821906 138822108 203 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Clec3b C-type lectin domain family 3, member b [ Mus musculus (house mouse) ] Gene ID: 21922, updated on 12-Aug-2019 Gene summary Official Symbol Clec3b provided by MGI Official Full Name C-type lectin domain family 3, member b provided by MGI Primary source MGI:MGI:104540 See related Ensembl:ENSMUSG00000025784 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 Tna Expression Biased expression in bladder adult (RPKM 244.6), mammary gland adult (RPKM 136.3) and 11 other tissues See more Orthologs human all Genomic context Location: 9 F4; 9 73.91 cM See Clec3b in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (123150946..123157432) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (123060064..123066550) Chromosome 9 - NC_000075.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Clec3b ENSMUSG00000025784 Description C-type lectin domain family 3, member b [Source:MGI Symbol;Acc:MGI:104540] Gene Synonyms Tna Location Chromosome 9: 123,150,946-123,157,432 forward strand. GRCm38:CM001002.2 About this gene This gene has 1 transcript (splice variant), 237 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Clec3b-201 ENSMUST00000026890.5 992 202aa ENSMUSP00000026890.4 Protein coding CCDS23656 Q8CFZ6 TSL:1 GENCODE basic APPRIS P1 26.49 kb Forward strand 123.145Mb 123.150Mb 123.155Mb 123.160Mb 123.165Mb Genes Clec3b-201 >protein coding Gm23323-201 >snoRNA (Comprehensive set... Contigs AC134248.3 > Regulatory Build 123.145Mb 123.150Mb 123.155Mb 123.160Mb 123.165Mb Reverse strand 26.49 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000026890 6.49 kb Forward strand Clec3b-201 >protein coding ENSMUSP00000026... Low complexity (Seg) Coiled-coils (Ncoils) Cleavage site (Sign... Superfamily SSF57944 C-type lectin fold SMART C-type lectin-like Prints PR01504 Pfam C-type lectin-like PROSITE profiles C-type lectin-like PROSITE patterns C-type lectin, conserved site PANTHER PTHR22799 PTHR22799:SF3 Gene3D C-type lectin-like/link domain superfamily CDD cd03596 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 202 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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