Mouse Fnta Conditional Knockout Project (CRISPR/Cas9)

https://www.alphaknockout.com Mouse Fnta Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Fnta conditional knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fnta gene (NCBI Reference Sequence: NM_008033 ; Ensembl: ENSMUSG00000015994 ) is located on Mouse chromosome 8. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000016138). 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 Fnta gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-12C10 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 25.38% 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: 2265 bp, and the size of intron 3 for 3'-loxP site insertion: 1320 bp. The size of effective cKO region: ~615 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 4 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Fnta Homology arm cKO region loxP site

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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. 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

Summary: Full Length(7115bp) | A(29.16% 2075) | C(20.04% 1426) | T(30.08% 2140) | G(20.72% 1474)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr8 - 26011455 26014454 3000 browser details YourSeq 121 553 716 3000 88.2% chr4 + 136250771 136251030 260 browser details YourSeq 108 554 703 3000 89.4% chr10 - 80460031 80460178 148 browser details YourSeq 107 555 826 3000 78.9% chr1 - 127686057 127686232 176 browser details YourSeq 105 566 725 3000 87.5% chr14 - 84269842 84270046 205 browser details YourSeq 103 554 712 3000 90.0% chr1 - 43952477 43952641 165 browser details YourSeq 97 557 828 3000 77.5% chr3 + 108161973 108162121 149 browser details YourSeq 96 553 724 3000 85.8% chr12 + 17531458 17531613 156 browser details YourSeq 95 553 687 3000 89.9% chr4 + 147819802 147819940 139 browser details YourSeq 95 573 833 3000 90.0% chr4 + 135852561 135852917 357 browser details YourSeq 91 523 664 3000 90.1% chrX - 73987709 73987855 147 browser details YourSeq 91 553 690 3000 88.8% chr1 - 133649700 133649837 138 browser details YourSeq 91 555 758 3000 78.9% chr7 + 29143459 29143644 186 browser details YourSeq 90 553 715 3000 91.8% chr17 - 83345580 83345843 264 browser details YourSeq 88 552 690 3000 89.3% chr17 + 80197146 80197296 151 browser details YourSeq 87 385 687 3000 75.3% chr10 - 75846978 75847141 164 browser details YourSeq 87 553 714 3000 84.5% chr3 + 58568185 58568334 150 browser details YourSeq 86 316 695 3000 89.9% chr16 - 37001172 37001683 512 browser details YourSeq 86 557 835 3000 75.3% chr6 + 39321282 39321432 151 browser details YourSeq 86 553 714 3000 83.1% chr12 + 52563389 52563537 149

Note: The 3000 bp section upstream of Exon 3 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% chr8 - 26007840 26010839 3000 browser details YourSeq 124 1433 1811 3000 89.3% chr7 + 88927734 88928157 424 browser details YourSeq 124 1435 1591 3000 93.2% chr1 + 170851689 170851855 167 browser details YourSeq 121 1433 1558 3000 98.5% chr4 - 107723732 107723858 127 browser details YourSeq 120 1410 1558 3000 94.7% chr16 - 4008262 4008733 472 browser details YourSeq 119 1433 1558 3000 97.7% chr2 + 137819104 137819230 127 browser details YourSeq 116 1437 1569 3000 96.1% chrX - 6841525 6841666 142 browser details YourSeq 116 1433 1568 3000 94.7% chr5 - 126073432 126073578 147 browser details YourSeq 114 1433 1570 3000 92.6% chr15 - 19477457 19477603 147 browser details YourSeq 114 1295 1558 3000 91.9% chr5 + 40371088 40371667 580 browser details YourSeq 113 1433 1558 3000 96.0% chr9 - 96903760 96903888 129 browser details YourSeq 113 1433 1587 3000 84.1% chr19 - 46427174 46427321 148 browser details YourSeq 113 1433 1558 3000 92.8% chr2 + 131110553 131110676 124 browser details YourSeq 113 1433 1909 3000 95.2% chr16 + 58581223 58581789 567 browser details YourSeq 112 1433 1575 3000 90.2% chrX - 135989518 135989672 155 browser details YourSeq 112 1433 1558 3000 98.3% chr8 - 113530373 113530504 132 browser details YourSeq 112 1435 1558 3000 96.0% chr8 - 46227846 46227975 130 browser details YourSeq 112 1434 1568 3000 91.8% chr6 - 9612249 9612383 135 browser details YourSeq 112 1433 1555 3000 96.7% chr12 - 32297131 32297253 123 browser details YourSeq 111 1433 1558 3000 95.9% chr5 - 151081458 151081584 127

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: Fnta farnesyltransferase, CAAX box, alpha [ Mus musculus (house mouse) ] Gene ID: 14272, updated on 12-Aug-2019

Gene summary

Official Symbol Fnta provided by MGI Official Full Name farnesyltransferase, CAAX box, alpha provided by MGI Primary source MGI:MGI:104683 See related Ensembl:ENSMUSG00000015994 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 FTA Expression Ubiquitous expression in placenta adult (RPKM 19.6), bladder adult (RPKM 17.6) and 28 other tissues See more Orthologs human all

Genomic context

Location: 8 A2; 8 14.27 cM See Fnta in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (25998688..26015610, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (27109194..27126073, complement)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 5 transcripts

Gene: Fnta ENSMUSG00000015994

Description farnesyltransferase, CAAX box, alpha [Source:MGI Symbol;Acc:MGI:104683] Gene Synonyms FTA Location Chromosome 8: 25,998,722-26,015,650 reverse strand. GRCm38:CM001001.2 About this gene This gene has 5 transcripts (splice variants), 217 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fnta-201 ENSMUST00000016138.10 1930 377aa ENSMUSP00000016138.9 Protein coding CCDS22205 Q541Z2 Q61239 TSL:1 GENCODE basic APPRIS P1

Fnta-203 ENSMUST00000209664.1 966 No protein - Retained intron - - TSL:3

Fnta-205 ENSMUST00000211718.1 950 No protein - Retained intron - - TSL:2

Fnta-204 ENSMUST00000209860.1 806 No protein - Retained intron - - TSL:2

Fnta-202 ENSMUST00000209247.1 635 No protein - Retained intron - - TSL:2

36.93 kb Forward strand

25.99Mb 26.00Mb 26.01Mb 26.02Mb Genes Gm45411-201 >lncRNA (Comprehensive set...

Contigs < AC093366.9 Genes (Comprehensive set... < Pomk-201protein coding < Fnta-201protein coding < Hook3-201protein coding

< Fnta-204retained intron

< Fnta-205retained intron

< Fnta-203retained intron

< Fnta-202retained intron

Regulatory Build

25.99Mb 26.00Mb 26.01Mb 26.02Mb Reverse strand 36.93 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000016138

< Fnta-201protein coding

Reverse strand 16.93 kb

ENSMUSP00000016... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF48439

Pfam Protein prenyltransferase, alpha subunit PROSITE profiles Protein prenyltransferase, alpha subunit PANTHER PTHR11129:SF5

PTHR11129 Gene3D 1.25.40.120

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 377

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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