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Mouse Mtfr1l Conditional Knockout Project (CRISPR/Cas9)

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Mouse Mtfr1l Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Mtfr1l Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Mtfr1l conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mtfr1l gene (NCBI Reference Sequence: NM_001256112 ; Ensembl: ENSMUSG00000046671 ) is located on Mouse chromosome 4. 7 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000102550). Exon 4~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Mtfr1l gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-288M17 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 4 starts from about 13.96% of the coding region. The knockout of Exon 4~6 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 1354 bp, and the size of intron 6 for 3'-loxP site insertion: 2576 bp. The size of effective cKO region: ~2201 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 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Mtfr1l 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(8701bp) | A(24.37% 2120) | C(23.18% 2017) | T(28.27% 2460) | G(24.18% 2104) 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% chr4 - 134531040 134534039 3000 browser details YourSeq 63 2917 3000 3000 88.1% chr17 + 47103617 47103713 97 browser details YourSeq 59 2919 3000 3000 86.6% chr15 - 84228990 84229084 95 browser details YourSeq 57 2917 3000 3000 84.6% chr1 - 87734628 87734724 97 browser details YourSeq 56 2920 3000 3000 85.0% chr10 + 59960223 59960708 486 browser details YourSeq 54 2916 3000 3000 86.5% chr13 + 57767457 57767554 98 browser details YourSeq 54 2930 3000 3000 88.8% chr11 + 22049715 22049964 250 browser details YourSeq 49 2917 2970 3000 96.3% chr18 + 54481255 54481321 67 browser details YourSeq 48 2917 2974 3000 93.2% chr17 - 86596320 86596758 439 browser details YourSeq 48 2932 2999 3000 85.3% chr12 - 110745326 110745393 68 browser details YourSeq 48 2940 3000 3000 90.2% chr1 + 150981201 150981565 365 browser details YourSeq 47 2917 2972 3000 94.5% chr11 + 78710702 78710769 68 browser details YourSeq 46 2907 2964 3000 87.3% chr8 + 18859753 18859809 57 browser details YourSeq 46 2920 2970 3000 96.1% chr18 + 10978400 10978463 64 browser details YourSeq 45 2918 2993 3000 92.6% chr10 - 61729793 61729882 90 browser details YourSeq 45 2929 2991 3000 85.8% chr16 + 57151020 57151082 63 browser details YourSeq 45 2917 2968 3000 94.3% chr11 + 120215224 120215292 69 browser details YourSeq 44 2917 2970 3000 92.6% chr18 - 69511977 69512043 67 browser details YourSeq 44 2945 3000 3000 89.3% chr1 + 165309602 165309657 56 browser details YourSeq 43 2932 3000 3000 81.2% chr19 + 49895973 49896041 69 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% chr4 - 134525839 134528838 3000 browser details YourSeq 405 1692 2162 3000 94.0% chr10 - 72114574 72115062 489 browser details YourSeq 325 2151 2542 3000 93.4% chr10 + 72114169 72114549 381 browser details YourSeq 313 824 1394 3000 93.4% chr1 - 59724372 60167578 443207 browser details YourSeq 291 841 1382 3000 95.1% chr4 + 44304951 44305552 602 browser details YourSeq 290 825 1391 3000 89.6% chr17 + 28566004 28566376 373 browser details YourSeq 288 833 1365 3000 94.5% chr4 - 135836636 135837217 582 browser details YourSeq 281 840 1398 3000 89.1% chr12 - 69147589 69148012 424 browser details YourSeq 270 838 1391 3000 88.6% chr6 + 125183191 125183719 529 browser details YourSeq 263 861 1391 3000 86.8% chr2 + 71410329 71410695 367 browser details YourSeq 261 835 1398 3000 86.4% chr19 - 9905851 9906261 411 browser details YourSeq 259 861 1392 3000 90.9% chr2 + 27517836 27518391 556 browser details YourSeq 257 853 1381 3000 86.9% chrX - 157456299 157456604 306 browser details YourSeq 252 842 1365 3000 92.6% chr6 + 115725853 115726393 541 browser details YourSeq 249 825 1400 3000 89.2% chr15 - 55303650 55304024 375 browser details YourSeq 239 474 1030 3000 88.6% chr18 - 36712450 36712891 442 browser details YourSeq 234 840 1316 3000 93.7% chr14 + 63084183 63084821 639 browser details YourSeq 232 828 1382 3000 87.6% chr15 + 81666708 81667022 315 browser details YourSeq 227 550 1033 3000 87.6% chr18 - 74963561 74963823 263 browser details YourSeq 225 835 1330 3000 92.0% chr17 - 31244341 31244985 645 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: Mtfr1l mitochondrial fission regulator 1-like [ Mus musculus (house mouse) ] Gene ID: 76824, updated on 24-Oct-2019 Gene summary Official Symbol Mtfr1l provided by MGI Official Full Name mitochondrial fission regulator 1-like provided by MGI Primary source MGI:MGI:1924074 See related Ensembl:ENSMUSG00000046671 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 Fam54b; AI480750; 2410166I05Rik Expression Ubiquitous expression in testis adult (RPKM 60.2), heart adult (RPKM 44.5) and 28 other tissues See more Orthologs human all Genomic context Location: 4; 4 D3 See Mtfr1l in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (134525555..134535387, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (134081477..134091174, complement) Chromosome 4 - NC_000070.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Mtfr1l ENSMUSG00000046671 Description mitochondrial fission regulator 1-like [Source:MGI Symbol;Acc:MGI:1924074] Gene Synonyms 2410166I05Rik, Fam54b Location Chromosome 4: 134,525,550-134,535,387 reverse strand. GRCm38:CM000997.2 About this gene This gene has 7 transcripts (splice variants), 200 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Mtfr1l- ENSMUST00000102550.9 2025 289aa ENSMUSP00000099609.3 Protein coding CCDS18775 Q9CWE0 TSL:1 201 GENCODE basic APPRIS P1 Mtfr1l- ENSMUST00000116279.9 1969 289aa ENSMUSP00000111983.3 Protein coding CCDS18775 Q9CWE0 TSL:1 202 GENCODE basic APPRIS P1 Mtfr1l- ENSMUST00000154769.7 1014 237aa ENSMUSP00000117943.1 Protein coding - D3YW54 CDS 3' 207 incomplete TSL:5 Mtfr1l- ENSMUST00000146808.1 925 225aa ENSMUSP00000120200.1 Protein coding - D3Z0X8 CDS 3' 206 incomplete TSL:2 Mtfr1l- ENSMUST00000131613.7 1768 202aa ENSMUSP00000123326.1 Nonsense mediated - D6RCX5 TSL:5 205 decay Mtfr1l- ENSMUST00000130187.7 4085 No - Retained intron - - TSL:2 204 protein Mtfr1l- ENSMUST00000129836.1 1048 No - Retained intron - - TSL:5 203 protein Page 6 of 8 https://www.alphaknockout.com 29.84 kb Forward strand 134.52Mb 134.53Mb 134.54Mb Genes Aunip-201 >protein coding (Comprehensive set... Contigs AL669982.10 > Genes (Comprehensive set... < Mtfr1l-201protein coding < Selenon-201protein coding < Mtfr1l-202protein coding < Selenon-202lncRNA < Mtfr1l-205nonsense mediated decay < Mtfr1l-204retained intron < Mtfr1l-207protein coding < Mtfr1l-206protein coding < Mtfr1l-203retained intron Regulatory Build 134.52Mb 134.53Mb 134.54Mb Reverse strand 29.84 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 RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000102550 < Mtfr1l-201protein coding Reverse strand 9.72 kb ENSMUSP00000099... Low complexity (Seg) Pfam Mitochondrial fission regulator 1 PANTHER Mitochondrial fission regulator 1 PTHR14215:SF3 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 289 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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