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Mouse Mcfd2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mcfd2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mcfd2 gene (NCBI Reference Sequence: NM_139295 ; Ensembl: ENSMUSG00000024150 ) is located on Mouse chromosome 17. 4 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 4 (Transcript: ENSMUST00000024963). 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 Mcfd2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-100H2 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 a null allele exhibit decreased serum factor V and VIII and aspartate transaminase serum levels with accumulation of the proteins in the ER of hepatocytes.

Exon 3 starts from about 33.79% 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: 621 bp, and the size of intron 3 for 3'-loxP site insertion: 1089 bp. The size of effective cKO region: ~714 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 2 3 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Mcfd2 Homology arm cKO region loxP site

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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(7160bp) | A(22.07% 1580) | C(25.15% 1801) | T(27.35% 1958) | G(25.43% 1821)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr17 - 87257557 87260556 3000 browser details YourSeq 678 1 958 3000 88.1% chr4 - 98748955 98750035 1081 browser details YourSeq 673 1 958 3000 87.2% chr1 - 193285433 193286410 978 browser details YourSeq 673 1 962 3000 88.3% chrX + 95029650 95030622 973 browser details YourSeq 672 1 964 3000 88.5% chr7 + 105666716 105667716 1001 browser details YourSeq 671 1 962 3000 88.0% chr9 + 81427704 81428706 1003 browser details YourSeq 667 1 958 3000 87.9% chr6 + 52335055 52336051 997 browser details YourSeq 660 1 960 3000 88.3% chr2 + 31773800 31774791 992 browser details YourSeq 659 1 958 3000 88.0% chr7 + 29738173 29739148 976 browser details YourSeq 658 1 958 3000 87.4% chr9 - 10320202 10321193 992 browser details YourSeq 657 1 958 3000 87.8% chr18 + 3641182 3642182 1001 browser details YourSeq 656 1 958 3000 87.3% chr6 - 97783277 97784272 996 browser details YourSeq 654 1 958 3000 87.7% chr10 - 77804730 77805712 983 browser details YourSeq 652 1 958 3000 87.3% chr9 + 57347915 57348910 996 browser details YourSeq 652 1 958 3000 87.7% chr6 + 54824007 54824984 978 browser details YourSeq 649 1 958 3000 87.7% chr11 - 3617484 3618475 992 browser details YourSeq 648 1 958 3000 87.7% chr9 - 77972804 77973797 994 browser details YourSeq 646 1 958 3000 87.2% chr6 - 3135460 3136451 992 browser details YourSeq 644 1 958 3000 86.5% chr10 - 40402088 40403087 1000 browser details YourSeq 643 1 963 3000 87.5% chr14 + 10271529 10272527 999

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% chr17 - 87253897 87256896 3000 browser details YourSeq 29 2116 2158 3000 94.0% chr10 - 63530326 63530371 46 browser details YourSeq 23 1860 1882 3000 100.0% chr19 + 3695041 3695063 23 browser details YourSeq 22 1110 1131 3000 100.0% chr4 - 55056836 55056857 22 browser details YourSeq 22 146 168 3000 100.0% chr11 + 105308748 105308772 25 browser details YourSeq 20 1827 1862 3000 77.8% chr2 + 50051461 50051496 36

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Mcfd2 multiple coagulation factor deficiency 2 [ Mus musculus (house mouse) ] Gene ID: 193813, updated on 12-Aug-2019

Gene summary

Official Symbol Mcfd2 provided by MGI Official Full Name multiple coagulation factor deficiency 2 provided by MGI Primary source MGI:MGI:2183439 See related Ensembl:ENSMUSG00000024150 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 F5f8d; Sdnsf; Lman1ip; 1810021C21Rik Expression Ubiquitous expression in liver E18 (RPKM 52.3), liver adult (RPKM 47.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 17; 17 E4 See Mcfd2 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (87254443..87265947, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (87653783..87665287, complement)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 7 transcripts

Gene: Mcfd2 ENSMUSG00000024150

Description multiple coagulation factor deficiency 2 [Source:MGI Symbol;Acc:MGI:2183439] Gene Synonyms 1810021C21Rik, F5F8D, LMAN1IP Location Chromosome 17: 87,254,443-87,265,935 reverse strand. GRCm38:CM001010.2 About this gene This gene has 7 transcripts (splice variants), 211 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mcfd2- ENSMUST00000024963.10 2038 145aa ENSMUSP00000024963.3 Protein CCDS29015 D0EW11 TSL:1 201 coding Q8K5B2 GENCODE basic APPRIS P1

Mcfd2- ENSMUST00000144236.8 2017 145aa ENSMUSP00000121820.1 Protein CCDS29015 D0EW11 TSL:1 204 coding Q8K5B2 GENCODE basic APPRIS P1

Mcfd2- ENSMUST00000129616.7 813 145aa ENSMUSP00000123352.1 Protein CCDS29015 D0EW11 TSL:3 202 coding Q8K5B2 GENCODE basic APPRIS P1

Mcfd2- ENSMUST00000145895.7 751 120aa ENSMUSP00000117105.1 Protein - D3Z6A4 TSL:2 205 coding GENCODE basic

Mcfd2- ENSMUST00000151155.7 603 131aa ENSMUSP00000119856.1 Protein - D3Z1G3 CDS 3' 206 coding incomplete TSL:5

Mcfd2- ENSMUST00000155904.1 327 74aa ENSMUSP00000114354.1 Protein - D3YVL4 CDS 3' 207 coding incomplete TSL:2

Mcfd2- ENSMUST00000139258.1 353 No - lncRNA - - TSL:5 203 protein

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31.49 kb Forward strand 87.25Mb 87.26Mb 87.27Mb Contigs AC129309.4 > AC084383.1 > Genes (Comprehensive set... < Mcfd2-201protein coding < 4833418N02Rik-201lncRNA

< Mcfd2-204protein coding < 4833418N02Rik-202lncRNA

< Mcfd2-202protein coding < 4833418N02Rik-203lncRNA

< Mcfd2-206protein coding

< Mcfd2-205protein coding

< Mcfd2-207protein coding

< Mcfd2-203lncRNA

Regulatory Build

87.25Mb 87.26Mb 87.27Mb Reverse strand 31.49 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000024963

< Mcfd2-201protein coding

Reverse strand 11.49 kb

ENSMUSP00000024... Cleavage site (Sign... Superfamily EF-hand domain pair Pfam EF-hand domain PROSITE profiles EF-hand domain PROSITE patterns EF-Hand 1, calcium-binding site PANTHER PTHR23104

PTHR23104:SF10 Gene3D 1.10.238.10

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 145

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

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