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Mouse Ythdf3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ythdf3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ythdf3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ythdf3 conditional knockout mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ythdf3 gene (NCBI Reference Sequence: NM_172677 ; Ensembl: ENSMUSG00000047213 ) is located on mouse chromosome 3. 6 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 6 (Transcript: ENSMUST00000108346). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the mouse Ythdf3 gene. (gRNA sequences are shown on the next page). To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-170B8 and RP23-113G15 from the C57BL/6 library 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 3 starts from about 2.8% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 5531 bp, and the size of intron 3 for 3'-loxP site insertion: 13623 bp. The size of effective cKO region: ~586 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 3 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ythdf3 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 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Summary: Full Length(7086bp) | A(30.27% 2145) | C(14.93% 1058) | T(36.25% 2569) | G(18.54% 1314) 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) ACTIONS QUERY SCORE START END QSIZE IDENTITY CHRO STRAND START END SPAN --------------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% 3 + 16186229 16189228 3000 browser details YourSeq 31 79 132 3000 94.3% 2 + 44156041 44156096 56 browser details YourSeq 25 199 226 3000 96.3% 10 - 105207383 105207411 29 browser details YourSeq 24 2788 2815 3000 92.9% 2 + 28874005 28874032 28 browser details YourSeq 23 2686 2712 3000 88.0% 16 - 66814051 66814076 26 browser details YourSeq 23 190 215 3000 96.0% 11 - 31101745 31101770 26 Note: The 3000 bp section upstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) ACTIONS QUERY SCORE START END QSIZE IDENTITY CHRO STRAND START END SPAN --------------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% 3 + 16189815 16192814 3000 browser details YourSeq 44 1234 1393 3000 90.8% 12 - 70074423 70074582 160 browser details YourSeq 43 1224 1396 3000 90.4% 11 + 97538307 97538492 186 browser details YourSeq 38 1327 1396 3000 80.7% 9 + 110000911 110000981 71 browser details YourSeq 35 1348 1395 3000 92.7% 13 + 9364423 9364471 49 browser details YourSeq 33 2772 2855 3000 97.3% 13 + 95770106 95770442 337 browser details YourSeq 30 1366 1402 3000 94.2% 1 - 177414483 177414526 44 browser details YourSeq 28 601 641 3000 96.7% 3 + 7669146 7669186 41 browser details YourSeq 28 1365 1398 3000 91.2% 14 + 101850605 101850638 34 browser details YourSeq 27 1365 1395 3000 93.6% 7 - 117678861 117678891 31 browser details YourSeq 27 1365 1393 3000 96.6% 14 - 20800081 20800109 29 browser details YourSeq 27 1365 1395 3000 93.6% 12 - 86670861 86670891 31 browser details YourSeq 27 1365 1395 3000 93.6% 10 + 29336071 29336101 31 browser details YourSeq 27 1250 1286 3000 96.6% 10 + 28079433 28079470 38 browser details YourSeq 26 1365 1396 3000 90.7% 11 - 117403615 117403646 32 browser details YourSeq 26 1365 1396 3000 90.7% 10 - 90066740 90066771 32 browser details YourSeq 26 1365 1396 3000 90.7% 16 + 29852840 29852871 32 browser details YourSeq 26 1365 1396 3000 90.7% 15 + 79128941 79128972 32 browser details YourSeq 25 1365 1393 3000 93.2% 12 - 83841001 83841029 29 browser details YourSeq 25 1365 1395 3000 90.4% 10 - 77935526 77935556 31 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: Ythdf3 YTH domain family 3 [ Mus musculus (house mouse) ] Gene ID: 229096, updated on 28-May-2017 Gene summary Official Symbol Ythdf3 provided by MGI Official Full Name YTH domain family 3 provided by MGI Primary source MGI:MGI:1918850 See related Ensembl:ENSMUSG00000047213 Vega:OTTMUSG00000051093 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 9130022A11Rik Orthologs human all Genomic context Location: 3; 3 A1 See Ythdf3 in Genome Data Viewer Map Viewer Exon count: 7 Annotation release Status Assembly Chr Location 106 current GRCm38.p4 (GCF_000001635.24) 3 NC_000069.6 (16183141..16217037) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (16083183..16117037) Chromosome 3 - NC_000069.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Ythdf3 ENSMUSG00000047213 Description YTH domain family 3 [Source:MGI Symbol;Acc:MGI:1918850] Synonyms 9130022A11Rik Location Chromosome 3: 16,183,212-16,217,037 forward strand. GRCm38:CM000996.2 About this gene This gene has 4 transcripts (splice variants), 66 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt RefSeq Flags Ythdf3- ENSMUST00000108345.8 5102 585aa ENSMUSP00000103982 Protein coding CCDS50869 Q8BYK6 NM_001145919 TSL:1 201 NP_001139391 GENCODE basic APPRIS P1 Ythdf3- ENSMUST00000108346.4 4929 596aa ENSMUSP00000103983 Protein coding CCDS38396 Q8BYK6 NM_172677 TSL:1 202 NP_766265 GENCODE basic Ythdf3- ENSMUST00000191774.5 3196 589aa ENSMUSP00000141610 Protein coding - Q8BYK6 NR_027375 TSL:1 203 GENCODE basic Ythdf3- ENSMUST00000193598.4 723 No - Processed - - - TSL:5 204 protein transcript 53.83 kb Forward strand 16.18Mb 16.19Mb 16.20Mb 16.21Mb 16.22Mb Genes (Comprehensive set... Ythdf3-201 >protein coding Ythdf3-202 >protein coding Ythdf3-203 >protein coding Ythdf3-204 >processed transcript Contigs AC122053.3 > Regulatory Build 16.18Mb 16.19Mb 16.20Mb 16.21Mb 16.22Mb Reverse strand 53.83 kb Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Regulation Legend Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site CTCF Activity in epigenome - Inactive Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000108346 33.83 kb Forward strand Ythdf3-201 >protein coding ENSMUSP00000103... Low complexity (Seg) hmmpanther PTHR12357:SF9 PTHR12357 Superfamily domains SSF81995 Pfam domain YTH domain PROSITE profiles YTH domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend Missense variant Synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 585 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC, VectorBuilder. Page 7 of 7.
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