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Mouse Ift74 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ift74 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ift74 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ift74 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ift74 gene (NCBI Reference Sequence: NM_026319 ; Ensembl: ENSMUSG00000028576 ) is located on Mouse chromosome 4. 20 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 20 (Transcript: ENSMUST00000030311). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ift74 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-173G7 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: Mice homozygous for an ENU-induced mutation exhibit complex congenital heart disease associated with heterotaxy. Exon 5 starts from about 17.0% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2197 bp, and the size of intron 6 for 3'-loxP site insertion: 5364 bp. The size of effective cKO region: ~836 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 5' gRNA region gRNA region 3' 1 4 5 6 20 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ift74 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. 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 Sequence 12 Summary: Full Length(7336bp) | A(29.63% 2174) | C(18.69% 1371) | T(32.62% 2393) | G(19.06% 1398) 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% chr4 + 94623728 94626727 3000 browser details YourSeq 285 305 775 3000 88.4% chr9 + 74914653 75128462 213810 browser details YourSeq 245 147 775 3000 81.0% chr10 - 109454751 109455147 397 browser details YourSeq 240 383 771 3000 84.5% chrX - 77742911 77743292 382 browser details YourSeq 232 386 775 3000 88.3% chr9 + 35576115 35576515 401 browser details YourSeq 224 404 775 3000 89.3% chr2 - 159205113 159205512 400 browser details YourSeq 221 384 773 3000 89.4% chr4 - 9679548 9679942 395 browser details YourSeq 221 411 772 3000 87.9% chr1 - 43394360 43394731 372 browser details YourSeq 219 367 775 3000 82.8% chr15 + 89773167 89773563 397 browser details YourSeq 219 403 775 3000 88.9% chr13 + 62942113 62942501 389 browser details YourSeq 218 373 775 3000 86.3% chrX - 75039257 75039656 400 browser details YourSeq 218 384 775 3000 86.6% chr8 + 9794608 9795017 410 browser details YourSeq 217 392 771 3000 87.6% chr1 - 43582673 43583050 378 browser details YourSeq 209 419 766 3000 87.5% chr18 - 58650368 58650735 368 browser details YourSeq 208 402 775 3000 88.0% chr9 + 36981695 36982085 391 browser details YourSeq 205 383 744 3000 88.9% chr6 - 30892429 30892809 381 browser details YourSeq 205 146 775 3000 83.5% chr7 + 90515061 90515482 422 browser details YourSeq 205 401 775 3000 85.3% chr5 + 133985164 133985518 355 browser details YourSeq 204 370 753 3000 89.1% chrX - 53132814 53133204 391 browser details YourSeq 204 364 783 3000 86.5% chr4 - 57939230 57939656 427 Note: The 3000 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 3000 1 3000 3000 100.0% chr4 + 94627564 94630563 3000 browser details YourSeq 1313 1385 3000 3000 92.2% chr14 - 49841127 49940325 99199 browser details YourSeq 1304 1382 3000 3000 92.6% chr4 + 34168082 34258436 90355 browser details YourSeq 1259 913 3000 3000 91.0% chr11 - 16356244 16357803 1560 browser details YourSeq 1253 1382 3000 3000 93.0% chr2 - 11466128 11467574 1447 browser details YourSeq 1242 1382 3000 3000 92.6% chr2 + 154278478 154279926 1449 browser details YourSeq 1241 1382 3000 3000 92.7% chr13 + 113624627 113626074 1448 browser details YourSeq 1235 1384 3000 3000 92.6% chr11 + 34955715 34957161 1447 browser details YourSeq 1233 1382 3000 3000 92.5% chr6 + 117746588 117748033 1446 browser details YourSeq 1232 1382 3000 3000 92.4% chr3 - 126874815 126876267 1453 browser details YourSeq 1231 1382 3000 3000 92.3% chr13 + 57010120 57011566 1447 browser details YourSeq 1229 1382 3000 3000 92.3% chrX - 35941627 35943074 1448 browser details YourSeq 1229 1382 3000 3000 92.5% chr13 - 47491741 47493187 1447 browser details YourSeq 1226 1382 3000 3000 92.3% chr13 - 74625426 74626873 1448 browser details YourSeq 1225 1382 3000 3000 92.2% chr4 - 107528563 107529994 1432 browser details YourSeq 1222 1382 3000 3000 92.2% chr19 - 11561587 11563032 1446 browser details YourSeq 1220 1382 3000 3000 92.0% chr8 + 79755220 79756668 1449 browser details YourSeq 1220 1384 3000 3000 91.5% chr18 + 79163634 79165079 1446 browser details YourSeq 1220 1382 3000 3000 92.4% chr13 + 8580195 8581641 1447 browser details YourSeq 1220 1382 3000 3000 92.0% chr10 + 82308167 82309612 1446 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ift74 intraflagellar transport 74 [ Mus musculus (house mouse) ] Gene ID: 67694, updated on 12-Aug-2019 Gene summary Official Symbol Ift74 provided by MGI Official Full Name intraflagellar transport 74 provided by MGI Primary source MGI:MGI:1914944 See related Ensembl:ENSMUSG00000028576 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 Cmg1; Ccdc2; b2b796Clo; 1700029H06Rik Expression Biased expression in testis adult (RPKM 8.8), CNS E11.5 (RPKM 7.9) and 12 other tissues See more Orthologs human all Genomic context Location: 4; 4 C5 See Ift74 in Genome Data Viewer Exon count: 27 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (94603266..94693233) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (94281211..94359924) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Ift74 ENSMUSG00000028576 Description intraflagellar transport 74 [Source:MGI Symbol;Acc:MGI:1914944] Gene Synonyms 1700029H06Rik, Ccdc2, Cmg1, b2b796Clo Location Chromosome 4: 94,614,491-94,693,229 forward strand. GRCm38:CM000997.2 About this gene This gene has 3 transcripts (splice variants), 198 orthologues, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ift74-201 ENSMUST00000030311.10 2139 600aa ENSMUSP00000030311.4 Protein coding CCDS51232 Q8BKE9 TSL:5 GENCODE basic APPRIS P1 Ift74-202 ENSMUST00000107104.2 1713 388aa ENSMUSP00000102721.2 Protein coding CCDS71420 B1AWG1 TSL:2 GENCODE basic Ift74-203 ENSMUST00000152289.1 549 No protein - lncRNA - - TSL:2 98.74 kb Forward strand 94.62Mb 94.64Mb 94.66Mb 94.68Mb 94.70Mb Genes (Comprehensive set... Ift74-202 >protein coding Mir872-201 >miRNA Ift74-203 >lncRNA Ift74-201 >protein coding Contigs AL732597.10 > AL772277.13 > Genes < Lrrc19-202protein coding (Comprehensive set... < Lrrc19-201protein coding Regulatory Build 94.62Mb 94.64Mb 94.66Mb 94.68Mb 94.70Mb Reverse strand 98.74 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000030311 78.74 kb Forward strand Ift74-201 >protein coding ENSMUSP00000030... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) PANTHER Intraflagellar transport protein 74 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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