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Mouse Zfpm1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Zfpm1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Zfpm1 (NCBI Reference Sequence: NM_009569 ; Ensembl: ENSMUSG00000049577 ) is located on Mouse 8. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000054052). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Zfpm1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-93D6 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: Homozygous mutants have poorly vascularized yolk sacs and small, pale livers. Mutants die between embryonic days 10.5 and 12.5 with severe anemia associated with a block in megakaryocyte development.

Exon 4 starts from about 10.52% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 16058 bp, and the size of intron 4 for 3'-loxP site insertion: 8256 bp. The size of effective cKO region: ~634 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 4 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Zfpm1 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. 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(7134bp) | A(22.29% 1590) | C(27.17% 1938) | T(22.13% 1579) | G(28.41% 2027)

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% chr8 + 122320451 122323450 3000 browser details YourSeq 26 132 171 3000 89.3% chr17 + 10326900 10326938 39 browser details YourSeq 23 1551 1573 3000 100.0% chr9 - 63735290 63735312 23 browser details YourSeq 23 115 137 3000 100.0% chr4 - 152388165 152388187 23 browser details YourSeq 21 440 460 3000 100.0% chr10 - 76553401 76553421 21 browser details YourSeq 21 1440 1460 3000 100.0% chr15 + 87570746 87570766 21

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% chr8 + 122324085 122327084 3000 browser details YourSeq 64 1591 1659 3000 98.6% chr8 + 121235789 121235882 94 browser details YourSeq 56 1598 2141 3000 67.2% chr12 - 88622101 88622264 164 browser details YourSeq 49 1706 2882 3000 74.3% chr1 + 188813624 189203816 390193 browser details YourSeq 47 1597 1658 3000 78.0% chr12 - 110156025 110156074 50 browser details YourSeq 45 1706 1817 3000 72.1% chr17 - 42984238 42984348 111 browser details YourSeq 45 2854 2939 3000 90.6% chr12 + 108028829 108029031 203 browser details YourSeq 44 1304 1812 3000 64.8% chr15 - 87050094 87050476 383 browser details YourSeq 44 1678 1823 3000 62.3% chr9 + 118832197 118832339 143 browser details YourSeq 43 2723 2939 3000 95.8% chr10 + 79617376 79617607 232 browser details YourSeq 42 1757 1816 3000 93.8% chr16 - 22463226 22463285 60 browser details YourSeq 41 1591 2143 3000 57.5% chr18 - 54489396 54489547 152 browser details YourSeq 41 1758 1821 3000 83.7% chr11 - 49676763 49676825 63 browser details YourSeq 41 1593 1659 3000 97.7% chr1 + 172786433 172786587 155 browser details YourSeq 40 1599 2143 3000 52.2% chr12 - 86543058 86543105 48 browser details YourSeq 39 1758 1816 3000 84.4% chr12 + 80595679 80595736 58 browser details YourSeq 38 2735 2934 3000 93.1% chr4 - 136882751 136882965 215 browser details YourSeq 37 1751 1817 3000 91.0% chr1 - 153114032 153114098 67 browser details YourSeq 36 1739 1809 3000 95.0% chr3 - 34603403 34603474 72 browser details YourSeq 35 1597 1660 3000 82.5% chr12 - 29302438 29302497 60

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Zfpm1 zinc finger protein, multitype 1 [ Mus musculus (house mouse) ] Gene ID: 22761, updated on 12-Aug-2019

Gene summary

Official Symbol Zfpm1 provided by MGI Official Full Name zinc finger protein, multitype 1 provided by MGI Primary source MGI:MGI:1095400 See related Ensembl:ENSMUSG00000049577 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 FOG; Fog1 Expression Broad expression in duodenum adult (RPKM 90.0), small intestine adult (RPKM 68.4) and 20 other tissues See more Orthologs human all

Genomic context

Location: 8; 8 E1 See Zfpm1 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (122281856..122337862)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (124806041..124861147)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 5 transcripts

Gene: Zfpm1 ENSMUSG00000049577

Description zinc finger protein, multitype 1 [Source:MGI Symbol;Acc:MGI:1095400] Gene Synonyms Fog1, Friend of GATA-1 Location Chromosome 8: 122,282,141-122,337,251 forward strand. GRCm38:CM001001.2 About this gene This gene has 5 transcripts (splice variants), 170 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 44 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Zfpm1-201 ENSMUST00000054052.14 3390 995aa ENSMUSP00000058037.8 Protein coding CCDS22733 O35615 TSL:1 GENCODE basic APPRIS P1

Zfpm1-202 ENSMUST00000176690.1 865 No protein - lncRNA - - TSL:3

Zfpm1-205 ENSMUST00000212315.1 704 No protein - lncRNA - - TSL:5

Zfpm1-204 ENSMUST00000177356.1 559 No protein - lncRNA - - TSL:2

Zfpm1-203 ENSMUST00000176883.1 314 No protein - lncRNA - - TSL:3

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75.11 kb Forward strand

122.28Mb 122.30Mb 122.32Mb 122.34Mb Gm20388-201 >protein coding (Comprehensive set...

Zfp469-202 >protein codZinfpgm1-201 >protein coding Gm45353-201 >lncRNA

Zfp469-201 >protein coding Zfpm1-203 >lncRNA Zfpm1-204 >lncRNA

Zfpm1-202 >lncRNA

Zfpm1-205 >lncRNA

Contigs < AC121976.2 Regulatory Build

122.28Mb 122.30Mb 122.32Mb 122.34Mb Reverse strand 75.11 kb

Regulation Legend CTCF Enhancer 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: ENSMUST00000054052

55.11 kb Forward strand

Zfpm1-201 >protein coding

ENSMUSP00000058... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Zinc finger C2H2 superfamily SMART Zinc finger C2H2-type Pfam Zinc finger C2H2-type PF12874

PROSITE profiles Zinc finger CCHC FOG-type

Zinc finger C2H2-type PROSITE patterns Zinc finger C2H2-type PANTHER PTHR12958:SF4

FOG family Gene3D 3.30.160.60

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

Variant Legend

inframe insertion inframe deletion missense variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 995

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

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