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Mouse Cd2ap Conditional Knockout Project (CRISPR/Cas9)

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Mouse Cd2ap Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Cd2ap Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Cd2ap conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cd2ap gene (NCBI Reference Sequence: NM_009847 ; Ensembl: ENSMUSG00000061665 ) is located on Mouse chromosome 17. 18 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 18 (Transcript: ENSMUST00000024709). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cd2ap gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-340J5 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: Homozygotes for a targeted null mutation exhibit impaired immune function and die at 6 to 7 weeks of age from kidney failure associated with podocyte defects and mesangial cell hyperplasia. Heterozygotes develop glomerular changes around 9 months. Exon 4 starts from about 16.75% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 6523 bp, and the size of intron 4 for 3'-loxP site insertion: 4416 bp. The size of effective cKO region: ~601 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 gRNA region 5' gRNA region 3' 1 4 18 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Cd2ap Homology arm 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(7101bp) | A(26.21% 1861) | C(18.87% 1340) | T(33.73% 2395) | G(21.19% 1505) 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% chr17 - 42839053 42842052 3000 browser details YourSeq 172 433 634 3000 91.4% chr16 - 35419864 35420062 199 browser details YourSeq 171 428 619 3000 94.8% chr11 - 5833259 6152391 319133 browser details YourSeq 169 433 618 3000 96.2% chr7 - 28853597 28853785 189 browser details YourSeq 169 430 618 3000 95.7% chr1 - 155021600 155021797 198 browser details YourSeq 167 428 636 3000 88.4% chr11 - 95439886 95440086 201 browser details YourSeq 167 433 620 3000 95.2% chr11 + 78714419 78714611 193 browser details YourSeq 166 430 618 3000 95.7% chr15 - 102318995 102319185 191 browser details YourSeq 166 430 618 3000 94.7% chr9 + 101067651 101067843 193 browser details YourSeq 166 428 618 3000 92.6% chr10 + 121377436 121377625 190 browser details YourSeq 166 428 618 3000 93.0% chr10 + 117098126 117098313 188 browser details YourSeq 164 438 633 3000 90.0% chr9 - 89087264 89087453 190 browser details YourSeq 164 430 618 3000 96.1% chr13 - 106862709 106863078 370 browser details YourSeq 164 438 633 3000 90.0% chr9 + 88586264 88586453 190 browser details YourSeq 164 430 618 3000 91.6% chr8 + 47597892 47598069 178 browser details YourSeq 164 432 617 3000 95.1% chr7 + 12902330 12902516 187 browser details YourSeq 164 456 769 3000 87.4% chr3 + 94617651 94617873 223 browser details YourSeq 164 426 618 3000 90.6% chr11 + 79068723 79068912 190 browser details YourSeq 163 430 618 3000 91.6% chr3 - 67519968 67520148 181 browser details YourSeq 163 424 621 3000 90.4% chr3 - 41047451 41047641 191 Note: The 3000 bp section upstream of Exon 4 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% chr17 - 42835452 42838451 3000 browser details YourSeq 63 1833 2374 3000 68.0% chr9 + 31957341 31957462 122 browser details YourSeq 58 1836 2374 3000 61.5% chr1 - 46014404 46014517 114 browser details YourSeq 56 1833 2374 3000 65.2% chr4 + 124577736 124577852 117 browser details YourSeq 50 2256 2374 3000 93.3% chr1 - 90501264 90501417 154 browser details YourSeq 49 2616 2699 3000 85.6% chr4 + 123255902 123256211 310 browser details YourSeq 49 1833 2374 3000 60.7% chr11 + 70922208 70922323 116 browser details YourSeq 47 1865 2380 3000 56.6% chr10 - 63238269 63238356 88 browser details YourSeq 47 2619 2699 3000 92.6% chr11 + 88396145 88396234 90 browser details YourSeq 47 2648 2718 3000 79.4% chr11 + 49860276 49860341 66 browser details YourSeq 46 2614 2699 3000 87.1% chr10 - 124387645 124387737 93 browser details YourSeq 45 2619 2697 3000 80.9% chr8 - 94380626 94380709 84 browser details YourSeq 45 1833 2376 3000 58.9% chr1 + 67390976 67391094 119 browser details YourSeq 44 2644 2720 3000 75.8% chr11 - 72167271 72167342 72 browser details YourSeq 44 2643 2699 3000 83.7% chr1 - 80434682 80434736 55 browser details YourSeq 43 2644 2702 3000 86.5% chr11 - 55014763 55014821 59 browser details YourSeq 43 2645 2699 3000 89.1% chr7 + 118265835 118265889 55 browser details YourSeq 43 2641 2699 3000 86.5% chr17 + 46106591 46106649 59 browser details YourSeq 43 2647 2699 3000 90.6% chr15 + 12431587 12431639 53 browser details YourSeq 42 2616 2699 3000 80.9% chr10 - 78711188 78711280 93 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Cd2ap CD2-associated protein [ Mus musculus (house mouse) ] Gene ID: 12488, updated on 10-Oct-2019 Gene summary Official Symbol Cd2ap provided by MGI Official Full Name CD2-associated protein provided by MGI Primary source MGI:MGI:1330281 See related Ensembl:ENSMUSG00000061665 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Mets1; C78928; METS-1; AL024079 Summary This gene encodes a scaffolding molecule that regulates the actin cytoskeleton. The protein directly interacts with Expression filamentous actin and a variety of cell membrane proteins through multiple actin binding sites, SH3 domains, and a proline- rich region containing binding sites for SH3 domains. The cytoplasmic protein localizes to membrane ruffles, lipid rafts, and the leading edges of cells. It is implicated in dynamic actin remodeling and membrane trafficking that occurs during receptor endocytosis and cytokinesis. The mouse genome contains at least two pseudogenes located on chromosomes 9 and 17. [provided by RefSeq, Jul 2008] Orthologs Ubiquitous expression in bladder adult (RPKM 14.9), placenta adult (RPKM 10.1) and 28 other tissues See more human all Genomic context Location: 17; 17 B3 See Cd2ap in Genome Data Viewer Exon count: 21 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (42748887..42876707, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (42929900..43013373, complement) Chromosome 17 - NC_000083.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Cd2ap ENSMUSG00000061665 Description CD2-associated protein [Source:MGI Symbol;Acc:MGI:1330281] Gene Synonyms METS-1, Mets1 Location Chromosome 17: 42,792,951-42,876,665 reverse strand. GRCm38:CM001010.2 About this gene This gene has 6 transcripts (splice variants), 200 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 15 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cd2ap-201 ENSMUST00000024709.8 5364 637aa ENSMUSP00000024709.7 Protein coding CCDS50114 Q9JLQ0 TSL:1 GENCODE basic APPRIS P1 Cd2ap-205 ENSMUST00000233476.1 3157 532aa ENSMUSP00000156812.1 Protein coding - A0A3B2W812 GENCODE basic Cd2ap-206 ENSMUST00000233626.1 5610 No protein - Retained intron - - - Cd2ap-204 ENSMUST00000233350.1 4712 No protein - Retained intron - - - Cd2ap-202 ENSMUST00000233123.1 3798 No protein - Retained intron - - - Cd2ap-203 ENSMUST00000233195.1 1621 No protein - Retained intron - - - 103.72 kb Forward strand 42.80Mb 42.82Mb 42.84Mb 42.86Mb 42.88Mb Contigs AC111082.23 > Genes (Comprehensive set... < Cd2ap-201protein coding < Cd2ap-204retained intron < Cd2ap-203retained intron < Cd2ap-205protein coding < Cd2ap-202retained intron < Cd2ap-206retained intron Regulatory Build 42.80Mb 42.82Mb 42.84Mb 42.86Mb 42.88Mb Reverse strand 103.72 kb Regulation Legend CTCF Enhancer 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 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000024709 < Cd2ap-201protein coding Reverse strand 83.44 kb ENSMUSP00000024... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SH3-like domain superfamily SMART SH3 domain Prints SH3 domain PR00499 Pfam SH3 domain SH3 domain PROSITE profiles SH3 domain PANTHER PTHR14167 CD2-associated protein Gene3D 2.30.30.40 CDD CD2-associated protein, second SH3 domain CD2-associated protein, first SH3 domain CD2-associated protein, third SH3 domain All sequence SNPs/i..
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