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Mouse Zfp266 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Zfp266 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Zfp266 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Zfp266 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Zfp266 gene (NCBI Reference Sequence: NM_001082485 ; Ensembl: ENSMUSG00000060510 ) is located on Mouse chromosome 9. 9 exons are identified, with the ATG start codon in exon 4 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000174462). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Zfp266 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-82H7 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 6 starts from about 6.35% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 798 bp, and the size of intron 6 for 3'-loxP site insertion: 2930 bp. The size of effective cKO region: ~627 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 5 6 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Zfp266 Homology arm cKO region loxP site Page 2 of 8 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(7127bp) | A(23.9% 1703) | C(20.4% 1454) | T(34.36% 2449) | G(21.34% 1521) 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 8 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 - 20505429 20508428 3000 browser details YourSeq 41 703 787 3000 84.8% chr16 - 18075890 18075976 87 browser details YourSeq 40 685 745 3000 93.5% chr2 - 121132301 121132363 63 browser details YourSeq 32 765 806 3000 97.1% chr6 - 86908274 86908316 43 browser details YourSeq 31 2774 2830 3000 94.2% chr15 - 88965836 88965894 59 browser details YourSeq 31 2766 2803 3000 92.2% chr11 - 50813669 50813711 43 browser details YourSeq 29 711 745 3000 91.5% chr11 - 97503567 97503601 35 browser details YourSeq 28 797 855 3000 86.7% chr9 + 57234701 57234757 57 browser details YourSeq 28 2010 2046 3000 96.7% chr16 + 48394651 48394688 38 browser details YourSeq 27 2032 2092 3000 72.2% chr1 - 39666413 39666473 61 browser details YourSeq 26 773 812 3000 96.5% chr12 - 51836911 51836950 40 browser details YourSeq 26 714 745 3000 90.7% chr1 - 60404385 60404416 32 browser details YourSeq 26 606 640 3000 85.8% chr3 + 25865561 25865593 33 browser details YourSeq 25 225 257 3000 86.3% chr13 - 75358420 75358451 32 browser details YourSeq 25 708 742 3000 85.8% chr2 + 170186512 170186546 35 browser details YourSeq 25 2006 2040 3000 93.2% chr10 + 12113080 12113116 37 browser details YourSeq 24 760 787 3000 92.9% chr1 + 76095855 76095882 28 browser details YourSeq 22 564 589 3000 92.4% chr1 + 181675926 181675951 26 Note: The 3000 bp section upstream of Exon 6 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 - 20501802 20504801 3000 browser details YourSeq 166 798 1567 3000 79.5% chr7 + 73002144 73002781 638 browser details YourSeq 121 416 1152 3000 89.7% chr2 + 21414548 21415331 784 browser details YourSeq 118 1336 1690 3000 74.9% chr15 + 91222019 91222360 342 browser details YourSeq 101 806 1227 3000 85.6% chr14 - 58580820 58581308 489 browser details YourSeq 89 36 915 3000 73.2% chr3 + 116216395 116217065 671 browser details YourSeq 87 571 868 3000 73.1% chr12 + 32971534 32971806 273 browser details YourSeq 83 1033 1227 3000 83.5% chr3 + 92881688 92881909 222 browser details YourSeq 80 559 816 3000 76.2% chr7 + 129230378 129230598 221 browser details YourSeq 78 860 1153 3000 84.9% chrX - 166888997 166889301 305 browser details YourSeq 75 1528 1754 3000 80.3% chr8 + 86413947 86414155 209 browser details YourSeq 72 615 915 3000 82.2% chrX + 68241967 68242246 280 browser details YourSeq 71 395 655 3000 75.6% chr18 - 19674271 19674445 175 browser details YourSeq 69 1049 1792 3000 85.0% chr1 - 12900489 12916328 15840 browser details YourSeq 68 1047 1270 3000 87.8% chr8 + 76578322 76578585 264 browser details YourSeq 67 1047 1227 3000 85.5% chr3 + 89806195 89806403 209 browser details YourSeq 67 657 906 3000 70.1% chr17 + 47660799 47660988 190 browser details YourSeq 65 637 874 3000 81.4% chr10 + 116118069 116118287 219 browser details YourSeq 64 430 631 3000 81.0% chr5 + 42019545 42019807 263 browser details YourSeq 62 436 1065 3000 90.5% chr9 - 123847314 123847942 629 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Zfp266 zinc finger protein 266 [ Mus musculus (house mouse) ] Gene ID: 77519, updated on 10-Oct-2019 Gene summary Official Symbol Zfp266 provided by MGI Official Full Name zinc finger protein 266 provided by MGI Primary source MGI:MGI:1924769 See related Ensembl:ENSMUSG00000060510 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 AW552317; 5330440G10Rik; 5730601F06Rik Expression Broad expression in CNS E11.5 (RPKM 3.9), CNS E14 (RPKM 2.8) and 22 other tissues See more Orthologs human all Genomic context Location: 9; 9 A3 See Zfp266 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (20495068..20521419, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (20299512..20325863, complement) Chromosome 9 - NC_000075.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Zfp266 ENSMUSG00000060510 Description zinc finger protein 266 [Source:MGI Symbol;Acc:MGI:1924769] Gene Synonyms 5330440G10Rik, 5730601F06Rik Location Chromosome 9: 20,495,068-20,521,417 reverse strand. GRCm38:CM001002.2 About this gene This gene has 6 transcripts (splice variants), 155 orthologues, 18 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 Zfp266-202 ENSMUST00000174462.7 6283 604aa ENSMUSP00000134217.1 Protein coding CCDS52732 E9Q2S7 TSL:1 GENCODE basic APPRIS P1 Zfp266-201 ENSMUST00000068296.7 2266 604aa ENSMUSP00000066012.7 Protein coding CCDS52732 E9Q2S7 TSL:1 GENCODE basic APPRIS P1 Zfp266-205 ENSMUST00000215908.1 894 99aa ENSMUSP00000149315.1 Protein coding - A0A1L1SR49 CDS 3' incomplete TSL:3 Zfp266-204 ENSMUST00000213418.1 485 11aa ENSMUSP00000150538.1 Protein coding - A0A1L1SU03 CDS 3' incomplete TSL:5 Zfp266-203 ENSMUST00000213275.1 679 No protein - lncRNA - - TSL:1 Zfp266-206 ENSMUST00000217488.1 368 No protein - lncRNA - - TSL:2 Page 6 of 8 https://www.alphaknockout.com 46.35 kb Forward strand 20.49Mb 20.50Mb 20.51Mb 20.52Mb 20.53Mb Contigs AC171206.2 > Genes (Comprehensive set... < Zfp426-210protein coding < Zfp266-202protein coding < Zfp426-202protein coding < Zfp266-201protein coding < Zfp426-213protein coding < Zfp266-205protein coding < Zfp426-201protein coding < Zfp266-204protein coding < Zfp426-203protein coding < Zfp266-206lncRNA < Zfp426-204nonsense mediated decay < Zfp266-203lncRNA < Zfp426-207protein coding < Zfp426-212protein coding < Zfp426-205protein coding Regulatory Build 20.49Mb 20.50Mb 20.51Mb 20.52Mb 20.53Mb Reverse strand 46.35 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000174462 < Zfp266-202protein coding Reverse strand 26.35 kb ENSMUSP00000134... Superfamily KRAB domain superfamily Zinc finger C2H2 superfamily SMART Krueppel-associated box Zinc finger C2H2-type Pfam Krueppel-associated box PF13894 Zinc finger C2H2-type PROSITE profiles Krueppel-associated box Zinc finger C2H2-type PROSITE patterns Zinc finger C2H2-type PANTHER PTHR24381 PTHR24381:SF265 Gene3D 3.30.160.60 CDD Krueppel-associated box All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 604 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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