https://www.alphaknockout.com

Mouse Nab2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Nab2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Nab2 (NCBI Reference Sequence: NM_008668 ; Ensembl: ENSMUSG00000025402 ) is located on Mouse 10. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000026469). Exon 2~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nab2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-124K20 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 null mice are viable and fertile with normal myelination.

Exon 2 starts from about 5.33% of the coding region. The knockout of Exon 2~6 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1237 bp, and the size of intron 6 for 3'-loxP site insertion: 951 bp. The size of effective cKO region: ~2908 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 2 3 4 5 6 7 22 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Nab2 cKO region Exon of mouse Stat6 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(9408bp) | A(22.53% 2120) | C(27.65% 2601) | T(20.48% 1927) | G(29.34% 2760)

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% chr10 - 127665389 127668388 3000 browser details YourSeq 28 2326 2356 3000 86.3% chr12 - 34189545 34189573 29 browser details YourSeq 26 2008 2037 3000 96.6% chr8 - 22664896 22664930 35 browser details YourSeq 24 1533 1562 3000 96.3% chr8 - 33961658 33961687 30 browser details YourSeq 24 1543 1571 3000 92.9% chr5 + 13182629 13182657 29 browser details YourSeq 23 1529 1554 3000 83.4% chr4 - 125850028 125850051 24 browser details YourSeq 22 2913 2934 3000 100.0% chr2 - 85384901 85384922 22 browser details YourSeq 22 1756 1777 3000 100.0% chr8 + 60875266 60875287 22

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% chr10 - 127659481 127662480 3000 browser details YourSeq 50 1077 1128 3000 100.0% chr11 + 65234307 65234516 210 browser details YourSeq 31 366 410 3000 71.5% chr1 - 97480503 97480537 35 browser details YourSeq 31 830 873 3000 86.1% chr17 + 21291320 21291376 57 browser details YourSeq 29 367 411 3000 70.0% chr5 - 76790705 76790734 30 browser details YourSeq 27 2386 2425 3000 96.6% chr15 + 95838506 95838547 42 browser details YourSeq 25 2220 2248 3000 96.5% chr4 - 153573816 153573853 38 browser details YourSeq 25 1130 1160 3000 96.3% chr6 + 52379156 52379188 33 browser details YourSeq 23 1106 1128 3000 100.0% chr12 - 45559565 45559587 23 browser details YourSeq 22 2912 2947 3000 80.6% chr10 - 28292891 28292926 36 browser details YourSeq 22 2905 2930 3000 92.4% chr1 + 165219156 165219181 26 browser details YourSeq 20 2839 2858 3000 100.0% chr4 - 96554007 96554026 20

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Nab2 Ngfi-A binding protein 2 [ Mus musculus (house mouse) ] Gene ID: 17937, updated on 12-Aug-2019

Gene summary

Official Symbol Nab2 provided by MGI Official Full Name Ngfi-A binding protein 2 provided by MGI Primary source MGI:MGI:107563 See related Ensembl:ENSMUSG00000025402 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 AI451907 Expression Ubiquitous expression in ovary adult (RPKM 34.3), thymus adult (RPKM 23.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 10 D3; 10 74.6 cM See Nab2 in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (127660918..127666703, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (127097974..127103759, complement)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 4 transcripts

Gene: Nab2 ENSMUSG00000025402

Description Ngfi-A binding protein 2 [Source:MGI Symbol;Acc:MGI:107563] Location Chromosome 10: 127,660,918-127,668,568 reverse strand. GRCm38:CM001003.2 About this gene This gene has 4 transcripts (splice variants), 183 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 27 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nab2-201 ENSMUST00000026469.8 2582 525aa ENSMUSP00000026469.2 Protein coding CCDS24247 Q61127 TSL:1 GENCODE basic APPRIS P3

Nab2-202 ENSMUST00000099157.3 2311 461aa ENSMUSP00000096761.3 Protein coding CCDS48719 Q3TYF1 TSL:1 GENCODE basic APPRIS ALT1

Nab2-203 ENSMUST00000128780.1 400 66aa ENSMUSP00000121737.1 Protein coding - D3Z292 CDS 3' incomplete TSL:3

Nab2-204 ENSMUST00000129252.1 386 46aa ENSMUSP00000118036.1 Protein coding - D3Z008 CDS 3' incomplete TSL:2

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27.65 kb Forward strand 127.655Mb 127.660Mb 127.665Mb 127.670Mb 127.675Mb Stat6-201 >protein coding Gm16229-201 >lncRNA Nemp1-202 >protein coding (Comprehensive set...

Stat6-203 >retained intron Stat6-204 >retained intron Nemp1-205 >lncRNA Nemp1-201 >protein coding

Stat6-205 >retained intron Nemp1-204 >lncRNA

Nemp1-203 >protein coding

Nemp1-206 >lncRNA

Contigs AC160970.7 > Genes (Comprehensive set... < Nab2-201protein coding < Gm16230-201lncRNA

< Nab2-202protein coding

< Nab2-203protein coding

< Nab2-204protein coding

Regulatory Build

127.655Mb 127.660Mb 127.665Mb 127.670Mb 127.675Mb Reverse strand 27.65 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000026469

< Nab2-201protein coding

Reverse strand 5.79 kb

ENSMUSP00000026... MobiDB lite Low complexity (Seg) Pfam Nab, N-terminal NAB co-repressor, domain

PANTHER NAB family

PTHR12623:SF6 Gene3D Sterile alpha motif/pointed domain superfamily NAB co-repressor domain 2 superfamily

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

Variant Legend

missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 525

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

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