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Mouse Nptx2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Nptx2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Nptx2 (NCBI Reference Sequence: NM_016789 ; Ensembl: ENSMUSG00000059991 ) is located on Mouse 5. 5 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 5 (Transcript: ENSMUST00000071782). 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 Nptx2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-286D4 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 mutation of this gene display a mild alteration in retinal ganglion cell innervation but are fertile with no obvious behavioral abnormalities.

Exon 2 starts from about 32.71% 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: 1635 bp, and the size of intron 2 for 3'-loxP site insertion: 5064 bp. The size of effective cKO region: ~717 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 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Nptx2 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(7217bp) | A(22.11% 1596) | C(25.52% 1842) | T(25.97% 1874) | G(26.4% 1905)

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% chr5 + 144544870 144547869 3000 browser details YourSeq 58 1365 1536 3000 84.6% chr11 - 119547227 119547437 211 browser details YourSeq 40 430 473 3000 90.7% chrY + 20602266 20602308 43 browser details YourSeq 32 2111 2227 3000 97.1% chr1 + 80595595 80595898 304 browser details YourSeq 28 2199 2226 3000 100.0% chr1 - 83570184 83570211 28 browser details YourSeq 22 2672 2693 3000 100.0% chr5 + 52362371 52362392 22 browser details YourSeq 20 1956 1975 3000 100.0% chr1 - 131042601 131042620 20 browser details YourSeq 20 2232 2251 3000 100.0% chr1 - 68759334 68759353 20 browser details YourSeq 20 2560 2579 3000 100.0% chr1 + 160350578 160350597 20

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% chr5 + 144548587 144551586 3000 browser details YourSeq 66 431 555 3000 83.4% chr2 - 135265830 135265942 113 browser details YourSeq 65 429 558 3000 94.6% chr11 + 106299512 106299800 289 browser details YourSeq 61 431 562 3000 95.6% chr10 - 60695363 60695615 253 browser details YourSeq 60 409 558 3000 92.7% chr5 - 118488531 118488819 289 browser details YourSeq 54 433 554 3000 80.0% chr12 + 51817516 51817611 96 browser details YourSeq 52 432 562 3000 89.1% chr10 - 69307601 69307755 155 browser details YourSeq 52 1389 1538 3000 78.8% chr1 + 171200831 171200969 139 browser details YourSeq 50 470 576 3000 84.5% chr1 + 33875966 33876068 103 browser details YourSeq 48 408 561 3000 96.2% chr12 + 83712700 83713186 487 browser details YourSeq 42 518 568 3000 89.4% chr10 - 41954945 41954993 49 browser details YourSeq 41 503 554 3000 91.9% chr3 - 87136306 87136361 56 browser details YourSeq 41 518 562 3000 95.6% chr18 - 33873279 33873323 45 browser details YourSeq 40 470 537 3000 79.5% chr5 - 132744899 132744966 68 browser details YourSeq 40 502 552 3000 93.5% chr1 - 84658205 84658255 51 browser details YourSeq 39 517 556 3000 100.0% chr7 - 120995089 120995129 41 browser details YourSeq 39 517 558 3000 97.7% chr1 - 189623949 189623991 43 browser details YourSeq 39 517 560 3000 95.5% chr8 + 95214759 95214803 45 browser details YourSeq 39 519 561 3000 97.7% chr7 + 79952550 79952599 50 browser details YourSeq 39 409 552 3000 64.8% chr10 + 111371194 111371291 98

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 information: Nptx2 neuronal pentraxin 2 [ Mus musculus (house mouse) ] Gene ID: 53324, updated on 10-Oct-2019

Gene summary

Official Symbol Nptx2 provided by MGI Official Full Name neuronal pentraxin 2 provided by MGI Primary source MGI:MGI:1858209 See related Ensembl:ENSMUSG00000059991 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 np2; Narp Expression Broad expression in cortex adult (RPKM 14.8), frontal lobe adult (RPKM 13.5) and 17 other tissues See more Orthologs human all

Genomic context

Location: 5 G2; 5 84.58 cM See Nptx2 in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (144545887..144557478)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (145306756..145318347)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 1 transcript

Gene: Nptx2 ENSMUSG00000059991

Description neuronal pentraxin 2 [Source:MGI Symbol;Acc:MGI:1858209] Gene Synonyms narp, np2 Location Chromosome 5: 144,545,902-144,557,478 forward strand. GRCm38:CM000998.2 About this gene This gene has 1 transcript (splice variant), 254 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nptx2-201 ENSMUST00000071782.7 2536 429aa ENSMUSP00000071687.6 Protein coding CCDS19849 O70340 TSL:1 GENCODE basic APPRIS P1

31.58 kb Forward strand

144.54Mb 144.55Mb 144.56Mb (Comprehensive set... Nptx2-201 >protein coding

Contigs < AC134523.5 AC164119.4 > Regulatory Build

144.54Mb 144.55Mb 144.56Mb Reverse strand 31.58 kb

Regulation Legend

CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

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Transcript: ENSMUST00000071782

11.58 kb Forward strand

Nptx2-201 >protein coding

ENSMUSP00000071... Low complexity (Seg) Coiled-coils (Ncoils) Cleavage site (Sign... Superfamily Concanavalin A-like lectin/glucanase domain superfamily

SMART Pentraxin-related

Prints Pentraxin-related Pfam Pentraxin-related

PROSITE profiles Pentraxin-related

PROSITE patterns Pentaxin, conserved site PANTHER PTHR19277

PTHR19277:SF1 Gene3D 2.60.120.200

CDD Pentraxin-related

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

Variant Legend

inframe insertion missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 429

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

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