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Mouse Inpp4a Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Inpp4a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Inpp4a gene (NCBI Reference Sequence: NM_001290797 ; Ensembl: ENSMUSG00000026113 ) is located on Mouse chromosome 1. 24 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 24 (Transcript: ENSMUST00000137266). Exon 14~15 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Inpp4a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-291B13 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: Homozygotes for a spontaneous mutation exhibit small size, ataxia, loss of cerebellar and hippocampal CA1 neurons, and death by 24 days of age from seizures and/or malnutrition.

Exon 14 starts from about 46.5% of the coding region. The knockout of Exon 14~15 will result in frameshift of the gene. The size of intron 13 for 5'-loxP site insertion: 1855 bp, and the size of intron 15 for 3'-loxP site insertion: 1079 bp. The size of effective cKO region: ~1837 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 13 14 15 24 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Inpp4a 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(8337bp) | A(23.04% 1921) | C(24.66% 2056) | T(27.22% 2269) | G(25.08% 2091)

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% chr1 + 37375592 37378591 3000

Note: The 3000 bp section upstream of Exon 14 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% chr1 + 37380429 37383428 3000 browser details YourSeq 98 1110 1358 3000 85.0% chr5 - 119914825 119915081 257 browser details YourSeq 98 1173 1391 3000 84.6% chr3 + 139057772 139058017 246 browser details YourSeq 98 1105 1376 3000 86.1% chr14 + 57885178 57885622 445 browser details YourSeq 96 802 1363 3000 74.7% chr9 + 105794195 105794568 374 browser details YourSeq 95 1105 1376 3000 88.0% chr3 + 96308918 96309302 385 browser details YourSeq 95 1086 1320 3000 85.0% chr2 + 147318427 147318659 233 browser details YourSeq 92 1080 1358 3000 83.4% chr3 - 129577494 129577796 303 browser details YourSeq 89 1244 1412 3000 73.8% chr11 - 106803209 106803371 163 browser details YourSeq 86 1181 1376 3000 80.4% chr5 - 113207636 113207829 194 browser details YourSeq 86 1105 1358 3000 87.7% chr13 + 37641295 37641549 255 browser details YourSeq 85 1104 1363 3000 89.7% chr9 - 35144336 35144597 262 browser details YourSeq 85 1086 1363 3000 76.1% chr1 + 36019798 36020094 297 browser details YourSeq 82 1186 1362 3000 75.3% chr5 + 125916557 125916701 145 browser details YourSeq 82 1187 1337 3000 88.7% chr13 + 34799727 34799896 170 browser details YourSeq 81 1083 1370 3000 87.3% chr10 - 118669212 119083205 413994 browser details YourSeq 80 1105 1363 3000 90.1% chr15 - 83472919 83473429 511 browser details YourSeq 79 1086 1363 3000 85.6% chr14 - 55277400 55277698 299 browser details YourSeq 79 1253 1412 3000 79.7% chr11 + 32125759 32125912 154 browser details YourSeq 78 1105 1412 3000 86.4% chr6 - 35533533 35533836 304

Note: The 3000 bp section downstream of Exon 15 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Inpp4a inositol polyphosphate-4-phosphatase, type I [ Mus musculus (house mouse) ] Gene ID: 269180, updated on 24-Oct-2019

Gene summary

Official Symbol Inpp4a provided by MGI Official Full Name inositol polyphosphate-4-phosphatase, type I provided by MGI Primary source MGI:MGI:1931123 See related Ensembl:ENSMUSG00000026113 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 107kDa; R74740; 9630012D15; D130048C09Rik Expression Ubiquitous expression in cerebellum adult (RPKM 12.1), frontal lobe adult (RPKM 10.4) and 26 other tissues See more Orthologs human all

Genomic context

Location: 1 B; 1 15.46 cM See Inpp4a in Genome Data Viewer

Exon count: 27

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (37299827..37410740)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (37356738..37467581)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 13 transcripts

Gene: Inpp4a ENSMUSG00000026113

Description inositol polyphosphate-4-phosphatase, type I [Source:MGI Symbol;Acc:MGI:1931123] Gene Synonyms 107kDa Location Chromosome 1: 37,299,865-37,410,736 forward strand. GRCm38:CM000994.2 About this gene This gene has 13 transcripts (splice variants), 257 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 20 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Inpp4a-201 ENSMUST00000027287.10 5691 939aa ENSMUSP00000027287.4 Protein coding CCDS35539 F6V2U0 TSL:5 GENCODE basic APPRIS P3

Inpp4a-203 ENSMUST00000114933.8 3004 690aa ENSMUSP00000110583.2 Protein coding CCDS35540 E9QAD0 TSL:1 GENCODE basic

Inpp4a-202 ENSMUST00000058307.14 2963 679aa ENSMUSP00000057233.8 Protein coding CCDS78570 A0A0R4J0Q2 TSL:1 GENCODE basic

Inpp4a-208 ENSMUST00000137266.7 2934 977aa ENSMUSP00000121803.1 Protein coding CCDS69879 D3Z230 TSL:5 GENCODE basic APPRIS ALT2

Inpp4a-209 ENSMUST00000140264.7 3676 890aa ENSMUSP00000121107.2 Protein coding - Q3UEQ1 TSL:1 GENCODE basic

Inpp4a-207 ENSMUST00000136846.7 3576 938aa ENSMUSP00000121518.2 Protein coding - E9Q9A0 TSL:5 GENCODE basic APPRIS ALT1

Inpp4a-205 ENSMUST00000132401.7 3234 972aa ENSMUSP00000123071.1 Protein coding - D3YUL8 TSL:5 GENCODE basic APPRIS ALT2

Inpp4a-206 ENSMUST00000132615.7 3038 954aa ENSMUSP00000115249.2 Protein coding - D3YUD3 TSL:5 GENCODE basic APPRIS ALT1

Inpp4a-211 ENSMUST00000168546.2 2964 679aa ENSMUSP00000130443.2 Protein coding - F6R4N2 TSL:5 GENCODE basic

Inpp4a-213 ENSMUST00000193774.5 1243 306aa ENSMUSP00000142118.1 Protein coding - A0A0A6YXS5 TSL:5 GENCODE basic

Inpp4a-212 ENSMUST00000193024.1 2874 No protein - Retained intron - - TSL:NA

Inpp4a-204 ENSMUST00000123124.1 463 No protein - Retained intron - - TSL:5

Inpp4a-210 ENSMUST00000146676.1 369 No protein - lncRNA - - TSL:3

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130.87 kb Forward strand 37.30Mb 37.35Mb 37.40Mb Genes (Comprehensive set... Inpp4a-210 >lncRNA Inpp4a-213 >protein coding

Inpp4a-201 >protein coding

Inpp4a-212 >retained intron Inpp4a-206 >protein coding

Inpp4a-209 >protein coding

Inpp4a-207 >protein coding

Inpp4a-205 >protein coding

Inpp4a-208 >protein coding

Inpp4a-203 >protein coding

Inpp4a-202 >protein coding

Inpp4a-211 >protein coding

Inpp4a-204 >retained intron

Contigs < AC133096.3 AC084390.1 > Genes < 1700074A21Rik-201TEC < 4930439A04Rik-201lncRNA < Coa5-201protein coding (Comprehensive set...

< Gm37135-201TEC

< 4930439A04Rik-202lncRNA

Regulatory Build

37.30Mb 37.35Mb 37.40Mb Reverse strand 130.87 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

RNA gene processed transcript

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Transcript: ENSMUST00000137266

50.34 kb Forward strand

Inpp4a-208 >protein coding

ENSMUSP00000121... Low complexity (Seg) Superfamily SSF49562 PANTHER PTHR12187:SF4

Inositol 3,4-bisphosphate 4-phosphatase Gene3D C2 domain superfamily

CDD cd04048

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 977

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

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