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Mouse Pogz Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Pogz gene (NCBI Reference Sequence: NM_172683 ; Ensembl: ENSMUSG00000038902 ) is located on Mouse chromosome 3. 19 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 19 (Transcript: ENSMUST00000107270). Exon 5~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pogz gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-336G12 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 lethality during organogenesis, fetal development and preweaning associated with fetal liver hypoplasia, small fetus size and anemia.

Exon 5 starts from about 10.88% of the coding region. The knockout of Exon 5~7 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1450 bp, and the size of intron 7 for 3'-loxP site insertion: 5412 bp. The size of effective cKO region: ~2746 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 5 6 7 19 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pogz 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(9246bp) | A(30.24% 2796) | C(20.13% 1861) | T(27.15% 2510) | G(22.49% 2079)

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% chr3 + 94859173 94862172 3000 browser details YourSeq 209 2117 2967 3000 93.8% chr3 - 9031545 9114485 82941 browser details YourSeq 206 2125 2979 3000 90.9% chr17 + 35029436 35494009 464574 browser details YourSeq 174 2094 2962 3000 92.7% chr13 - 55170081 55466950 296870 browser details YourSeq 157 2450 2971 3000 84.9% chr10 + 23123077 23123303 227 browser details YourSeq 138 2681 2967 3000 93.8% chr12 - 41125510 41125956 447 browser details YourSeq 137 2549 2976 3000 81.4% chr2 + 164324577 164324748 172 browser details YourSeq 137 2824 2986 3000 95.5% chr17 + 29067296 29067470 175 browser details YourSeq 136 2817 2976 3000 93.6% chr14 + 60108759 60108925 167 browser details YourSeq 134 2824 2978 3000 94.8% chr15 + 99805956 99806129 174 browser details YourSeq 134 2824 2978 3000 94.2% chr1 + 74614831 74615000 170 browser details YourSeq 133 2826 2976 3000 94.6% chr19 + 59079491 59079641 151 browser details YourSeq 132 2824 2976 3000 92.1% chr4 - 149681204 149681355 152 browser details YourSeq 132 2826 2976 3000 91.9% chr12 - 54770893 54771041 149 browser details YourSeq 131 2825 2979 3000 92.6% chr7 - 19819591 19819744 154 browser details YourSeq 131 2824 2978 3000 90.8% chr11 - 98914709 98914861 153 browser details YourSeq 131 2830 2977 3000 94.6% chr11 - 59845351 59845498 148 browser details YourSeq 131 2824 2979 3000 92.4% chr2 + 121284935 121285091 157 browser details YourSeq 131 2824 2978 3000 90.8% chr19 + 18834456 18834607 152 browser details YourSeq 131 2825 2977 3000 90.3% chr12 + 27890083 27890227 145

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% chr3 + 94864919 94867918 3000 browser details YourSeq 304 81 2929 3000 94.5% chr11 - 106306791 106482305 175515 browser details YourSeq 221 88 1431 3000 89.8% chr1 - 133159934 133507721 347788 browser details YourSeq 167 599 1452 3000 85.2% chr9 - 45921957 45922178 222 browser details YourSeq 150 2768 2937 3000 94.0% chr19 + 57545794 57545962 169 browser details YourSeq 147 824 1453 3000 82.1% chr18 + 6144417 6144663 247 browser details YourSeq 140 987 1149 3000 93.3% chr16 - 90286425 90286587 163 browser details YourSeq 139 985 1145 3000 93.8% chr19 - 8759329 8759491 163 browser details YourSeq 139 2779 2930 3000 96.0% chr18 - 75026312 75026463 152 browser details YourSeq 139 2771 2927 3000 94.9% chrX + 38574557 38574716 160 browser details YourSeq 138 987 1146 3000 93.7% chr2 - 90857969 90858128 160 browser details YourSeq 137 987 1143 3000 94.3% chr9 - 14481565 14481723 159 browser details YourSeq 137 987 1585 3000 81.2% chr18 - 17443710 17444066 357 browser details YourSeq 136 2771 2926 3000 94.2% chr9 + 103196850 103197008 159 browser details YourSeq 136 987 1149 3000 91.8% chr2 + 32003904 32004065 162 browser details YourSeq 136 990 1150 3000 92.5% chr10 + 81198545 81198705 161 browser details YourSeq 136 996 1150 3000 94.2% chr1 + 172401946 172402101 156 browser details YourSeq 135 987 1140 3000 95.4% chr5 - 97009372 97009534 163 browser details YourSeq 135 990 1149 3000 91.7% chr2 - 91815174 91815331 158 browser details YourSeq 135 996 1146 3000 95.4% chr11 - 84978772 84978925 154

Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Pogz pogo transposable element with ZNF domain [ Mus musculus (house mouse) ] Gene ID: 229584, updated on 10-Oct-2019

Gene summary

Official Symbol Pogz provided by MGI Official Full Name pogo transposable element with ZNF domain provided by MGI Primary source MGI:MGI:2442117 See related Ensembl:ENSMUSG00000038902 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 9530006B08Rik Expression Ubiquitous expression in whole brain E14.5 (RPKM 13.7), CNS E14 (RPKM 13.7) and 28 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 F2.1 See Pogz in Genome Data Viewer

Exon count: 21

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (94822989..94883577)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (94641489..94687491)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 10 transcripts

Gene: Pogz ENSMUSG00000038902

Description pogo transposable element with ZNF domain [Source:MGI Symbol;Acc:MGI:2442117] Gene Synonyms 9530006B08Rik Location Chromosome 3: 94,837,567-94,882,326 forward strand. GRCm38:CM000996.2 About this gene This gene has 10 transcripts (splice variants), 130 orthologues, 8 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pogz- ENSMUST00000107270.8 6399 1409aa ENSMUSP00000102891.2 Protein coding CCDS17596 B9EIG6 TSL:1 205 Q8BZH4 GENCODE basic APPRIS P3

Pogz- ENSMUST00000042402.11 4775 1400aa ENSMUSP00000037523.5 Protein coding CCDS50985 Q0VGT3 TSL:1 201 GENCODE basic APPRIS ALT2

Pogz- ENSMUST00000107269.1 5966 1314aa ENSMUSP00000102890.1 Protein coding - D3YUW8 TSL:5 204 GENCODE basic

Pogz- ENSMUST00000107266.7 4100 1356aa ENSMUSP00000102887.1 Protein coding - D3YUX1 TSL:5 203 GENCODE basic APPRIS ALT2

Pogz- ENSMUST00000140397.1 517 54aa ENSMUSP00000122492.1 Nonsense mediated - F7D0L1 CDS 5' 209 decay incomplete TSL:5

Pogz- ENSMUST00000126235.7 2933 No - Retained intron - - TSL:1 207 protein

Pogz- ENSMUST00000142253.1 777 No - Retained intron - - TSL:2 210 protein

Pogz- ENSMUST00000132544.1 744 No - Retained intron - - TSL:3 208 protein

Pogz- ENSMUST00000107265.7 742 No - lncRNA - - TSL:3 202 protein

Pogz- ENSMUST00000125638.1 279 No - lncRNA - - TSL:5 206 protein

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64.76 kb Forward strand

Genes (Comprehensive set... Gm15263-201 >processed pseudogene Pogz-208 >retained intron Pogz-210 >retained intron

Pogz-205 >protein coding

Pogz-202 >lncRNA Pogz-209 >nonsense mediated decay

Pogz-204 >protein coding

Pogz-201 >protein coding

Pogz-203 >protein coding

Pogz-207 >retained intron Pogz-206 >lncRNA

Contigs AC087903.27 > AC087062.25 > Genes < Psmb4-203retained intron (Comprehensive set...

< Psmb4-202retained intron

< Psmb4-204retained intron

< Psmb4-205retained intron

< Psmb4-201protein coding

Regulatory Build

Reverse strand 64.76 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene pseudogene

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Transcript: ENSMUST00000107270

44.76 kb Forward strand

Pogz-205 >protein coding

ENSMUSP00000102... MobiDB lite Low complexity (Seg) Superfamily Zinc finger C2H2 superfamily Homeobox-like domain superfamily

SMART Zinc finger C2H2-type HTH CenpB-type DNA-binding domain Pfam HTH CenpB-type DNA-binding domain

DDE superfamily endonuclease domain PROSITE profiles Zinc finger C2H2-type HTH CenpB-type DNA-binding domain PROSITE patterns Zinc finger C2H2-type PANTHER PTHR24403:SF59

PTHR24403 Gene3D 3.30.160.60 1.10.10.60

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

Variant Legend missense variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1409

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

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