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Mouse Pip4k2a Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Pip4k2a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Pip4k2a gene (NCBI Reference Sequence: NM_008845 ; Ensembl: ENSMUSG00000026737 ) is located on Mouse chromosome 2. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000006912). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pip4k2a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-346P16 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 are viable and appear phenotypically normal.

Exon 2 starts from about 11.93% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 89699 bp, and the size of intron 2 for 3'-loxP site insertion: 1729 bp. The size of effective cKO region: ~598 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 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pip4k2a 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(7098bp) | A(25.98% 1844) | C(22.67% 1609) | T(27.92% 1982) | G(23.43% 1663)

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% chr2 - 18908403 18911402 3000 browser details YourSeq 42 1982 2032 3000 95.7% chr11 - 98508390 98508443 54 browser details YourSeq 41 1982 2032 3000 93.4% chr7 - 98763284 98763335 52 browser details YourSeq 41 1982 2032 3000 79.6% chr12 - 100818582 100818625 44 browser details YourSeq 41 1982 2032 3000 93.4% chr12 - 100818903 100818953 51 browser details YourSeq 41 1982 2032 3000 79.6% chr10 - 9670099 9670142 44 browser details YourSeq 37 1982 2025 3000 95.0% chr7 - 98763220 98763266 47 browser details YourSeq 37 1982 2025 3000 82.1% chr17 - 15722872 15722910 39 browser details YourSeq 36 1982 2024 3000 94.9% chr17 - 15722703 15722747 45 browser details YourSeq 36 1982 2025 3000 95.0% chr11 + 80911605 80911651 47 browser details YourSeq 35 1986 2032 3000 92.5% chr9 - 118377591 118377637 47 browser details YourSeq 34 1984 2024 3000 81.1% chr12 - 77874255 77874291 37 browser details YourSeq 32 2599 2647 3000 83.0% chr15 + 77998896 77998946 51 browser details YourSeq 30 1498 1555 3000 87.5% chr6 + 39217991 39218046 56 browser details YourSeq 28 1982 2009 3000 100.0% chr10 + 26542355 26542382 28 browser details YourSeq 28 2615 2650 3000 91.2% chr10 + 12651646 12651683 38 browser details YourSeq 27 1982 2008 3000 100.0% chr12 - 100818531 100818557 27 browser details YourSeq 27 1985 2014 3000 96.7% chr18 + 84182904 84182934 31 browser details YourSeq 27 2632 2660 3000 96.6% chr1 + 89064838 89064866 29 browser details YourSeq 26 2605 2650 3000 78.3% chr10 + 18291778 18291823 46

Note: The 3000 bp section upstream of Exon 2 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% chr2 - 18904805 18907804 3000 browser details YourSeq 197 1930 2151 3000 96.3% chr7 - 83707554 83707933 380 browser details YourSeq 154 1940 2147 3000 89.6% chr7 - 78967909 78968092 184 browser details YourSeq 152 1939 2374 3000 91.2% chr1 + 172156480 172156952 473 browser details YourSeq 149 1933 2147 3000 86.3% chr1 + 157759479 157759658 180 browser details YourSeq 148 1951 2151 3000 89.4% chr14 - 57225500 57225663 164 browser details YourSeq 145 1955 2146 3000 90.6% chr5 - 38573305 38573469 165 browser details YourSeq 142 1967 2416 3000 78.5% chr15 + 74649494 74649937 444 browser details YourSeq 141 1953 2146 3000 89.2% chr6 - 119011290 119011461 172 browser details YourSeq 141 1954 2147 3000 88.6% chr1 - 169568454 169568617 164 browser details YourSeq 141 1957 2145 3000 88.7% chr19 + 8759328 8759489 162 browser details YourSeq 140 1932 2146 3000 86.2% chr8 - 111908278 111908468 191 browser details YourSeq 140 1954 2142 3000 87.9% chr14 - 20898444 20898603 160 browser details YourSeq 138 1967 2164 3000 87.9% chr4 - 137340077 137340251 175 browser details YourSeq 137 1958 2146 3000 89.2% chr13 - 67691270 67691442 173 browser details YourSeq 136 1962 2146 3000 88.6% chr4 - 126955690 126955845 156 browser details YourSeq 136 1953 2146 3000 86.2% chr18 + 58908862 58909024 163 browser details YourSeq 135 1958 2146 3000 87.7% chr16 - 4917335 4917494 160 browser details YourSeq 135 1933 2151 3000 87.9% chr15 - 37576443 37576610 168 browser details YourSeq 134 1952 2146 3000 85.0% chr15 - 81838295 81838456 162

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Pip4k2a phosphatidylinositol-5-phosphate 4-kinase, type II, alpha [ Mus musculus (house mouse) ] Gene ID: 18718, updated on 12-Aug-2019

Gene summary

Official Symbol Pip4k2a provided by MGI Official Full Name phosphatidylinositol-5-phosphate 4-kinase, type II, alpha provided by MGI Primary source MGI:MGI:1298206 See related Ensembl:ENSMUSG00000026737 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 Pip5k2a; AW742916 Expression Ubiquitous expression in thymus adult (RPKM 11.5), cerebellum adult (RPKM 9.3) and 24 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 A3 See Pip4k2a in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (18842255..18998291, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (18763883..18919748, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 3 transcripts

Gene: Pip4k2a ENSMUSG00000026737

Description phosphatidylinositol-5-phosphate 4-kinase, type II, alpha [Source:MGI Symbol;Acc:MGI:1298206] Gene Synonyms Pip5k2a Location Chromosome 2: 18,842,255-18,998,126 reverse strand. GRCm38:CM000995.2 About this gene This gene has 3 transcripts (splice variants), 210 orthologues, 6 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pip4k2a- ENSMUST00000006912.11 3477 405aa ENSMUSP00000006912.5 Protein CCDS15712 O70172 TSL:1 201 coding Q544E3 GENCODE basic APPRIS P1

Pip4k2a- ENSMUST00000152981.1 641 214aa ENSMUSP00000119075.1 Protein - F6RJE8 CDS 5' and 3' 203 coding incomplete TSL:3

Pip4k2a- ENSMUST00000148583.7 317 83aa ENSMUSP00000115475.1 Protein - A2ASS3 CDS 5' incomplete 202 coding TSL:5

175.87 kb Forward strand 18.85Mb 18.90Mb 18.95Mb 19.00Mb Genes 4930426L09Rik-201 >lncRNA (Comprehensive set...

4930426L09Rik-202 >lncRNA

Contigs AL928680.5 > AL928653.4 > AL844855.15 >

Genes (Comprehensive set... < Pip4k2a-201protein coding

< Pip4k2a-202protein coding

< Pip4k2a-203protein coding

Regulatory Build

18.85Mb 18.90Mb 18.95Mb 19.00Mb Reverse strand 175.87 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

RNA gene

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Transcript: ENSMUST00000006912

< Pip4k2a-201protein coding

Reverse strand 155.87 kb

ENSMUSP00000006... MobiDB lite Low complexity (Seg) Superfamily SSF56104

SMART Phosphatidylinositol-4-phosphate 5-kinase, core Pfam Phosphatidylinositol-4-phosphate 5-kinase, core

PROSITE profiles Phosphatidylinositol-4-phosphate 5-kinase, core

PANTHER PTHR23086:SF21

Phosphatidylinositol-4-phosphate 5-kinase Gene3D Phosphatidylinositol-4-phosphate 5-kinase, N-terminal Phosphatidylinositol-4-phosphate 5-kinase, C-terminal

CDD cd17309

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

Variant Legend

missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 405

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

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