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Mouse Atg9b Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Atg9b conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Atg9b (NCBI Reference Sequence: NM_001002897 ; Ensembl: ENSMUSG00000038295 ) is located on Mouse 5. 14 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000059401). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Atg9b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-237P14 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:

Exon 5 starts from about 29.61% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 539 bp, and the size of intron 5 for 3'-loxP site insertion: 1337 bp. The size of effective cKO region: ~642 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 4 5 6 7 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Atg9b 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(7142bp) | A(20.65% 1475) | C(27.23% 1945) | T(25.2% 1800) | G(26.91% 1922)

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 - 24390173 24393172 3000 browser details YourSeq 46 656 755 3000 92.8% chr13 + 106952899 106953002 104 browser details YourSeq 44 2277 2405 3000 88.0% chr19 - 27382247 27382424 178 browser details YourSeq 42 2237 2290 3000 88.9% chr10 + 62249099 62249152 54 browser details YourSeq 40 1389 1674 3000 95.6% chr11 - 59336352 59337139 788 browser details YourSeq 39 2237 2309 3000 95.4% chr3 - 58440769 58440841 73 browser details YourSeq 33 2278 2312 3000 97.2% chr7 - 105503477 105503511 35 browser details YourSeq 32 2277 2316 3000 90.0% chr5 + 139048923 139048962 40 browser details YourSeq 31 2277 2317 3000 87.9% chr19 - 42776029 42776069 41 browser details YourSeq 31 2277 2317 3000 87.9% chr5 + 100114153 100114193 41 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 89014564 89014596 33 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 88015549 88015581 33 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 87078699 87078731 33 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 82187044 82187076 33 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 81419824 81419856 33 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 77280819 77280851 33 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 75677054 75677086 33 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 74823874 74823906 33 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 73631179 73631211 33 browser details YourSeq 30 2367 2397 3000 100.0% chrY - 71877899 71877931 33

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% chr5 - 24386531 24389530 3000 browser details YourSeq 152 353 516 3000 96.4% chr14 - 11292777 11292940 164 browser details YourSeq 151 353 516 3000 96.4% chr17 - 28551892 28552056 165 browser details YourSeq 150 353 525 3000 91.2% chr6 + 140629038 140629207 170 browser details YourSeq 149 352 516 3000 95.2% chr8 - 83145902 83146066 165 browser details YourSeq 149 353 516 3000 95.8% chr6 - 29217672 29217837 166 browser details YourSeq 149 353 516 3000 95.8% chr1 - 63107176 63107342 167 browser details YourSeq 148 353 516 3000 95.2% chr5 - 135954722 135954885 164 browser details YourSeq 148 353 516 3000 95.2% chr13 - 67482998 67483161 164 browser details YourSeq 148 356 516 3000 96.3% chr17 + 46871670 46871831 162 browser details YourSeq 147 354 516 3000 95.1% chr2 + 157319638 157319800 163 browser details YourSeq 146 353 516 3000 94.6% chr11 + 23819493 23819656 164 browser details YourSeq 146 356 516 3000 95.7% chr10 + 81045227 81045398 172 browser details YourSeq 145 351 509 3000 93.6% chr5 + 90490943 90491098 156 browser details YourSeq 144 357 516 3000 95.7% chr13 - 58184635 58184820 186 browser details YourSeq 144 359 514 3000 94.9% chr12 - 81677601 81677755 155 browser details YourSeq 144 355 516 3000 94.5% chr9 + 53360844 53361005 162 browser details YourSeq 144 351 516 3000 93.4% chr12 + 66156287 66156452 166 browser details YourSeq 144 359 516 3000 95.6% chr11 + 107134303 107134460 158 browser details YourSeq 143 353 516 3000 94.0% chr9 - 65778207 65778371 165

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Atg9b related 9B [ Mus musculus (house mouse) ] Gene ID: 213948, updated on 12-Aug-2019

Gene summary

Official Symbol Atg9b provided by MGI Official Full Name autophagy related 9B provided by MGI Primary source MGI:MGI:2685420 See related Ensembl:ENSMUSG00000038295 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 eONE; sONE; Gm574; Apg912; Apg9l2; Apgdc2; Nos3as Expression Broad expression in stomach adult (RPKM 10.7), lung adult (RPKM 3.8) and 19 other tissues See more Orthologs all

Genomic context

Location: 5 A3; 5 11.49 cM See Atg9b in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (24384181..24392143, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (23889999..23897961, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 3 transcripts

Gene: Atg9b ENSMUSG00000038295

Description autophagy related 9B [Source:MGI Symbol;Acc:MGI:2685420] Gene Synonyms Apg9l2, LOC213948, Nos3as, eONE Location Chromosome 5: 24,384,181-24,392,143 reverse strand. GRCm38:CM000998.2 About this gene This gene has 3 transcripts (splice variants), 94 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Atg9b-201 ENSMUST00000059401.6 3902 922aa ENSMUSP00000051864.6 Protein coding CCDS39027 Q6EBV9 TSL:1 GENCODE basic APPRIS P1

Atg9b-203 ENSMUST00000138716.7 1837 No protein - lncRNA - - TSL:5

Atg9b-202 ENSMUST00000128831.1 490 No protein - lncRNA - - TSL:3

27.96 kb Forward strand

24.38Mb 24.39Mb 24.40Mb Nos3-201 >protein coding Abcb8-207 >protein coding (Comprehensive set...

Nos3-202 >protein coding Abcb8-203 >protein coding

Abcb8-201 >protein coding

Abcb8-202 >lncRNA

Abcb8-205 >retained intron

Abcb8-209 >protein coding

Contigs < AC113055.9 Genes (Comprehensive set... < Gm15587-201lncRNA < Atg9b-201protein coding

< Gm15587-202lncRNA < Atg9b-203lncRNA

< Atg9b-202lncRNA

Regulatory Build

24.38Mb 24.39Mb 24.40Mb Reverse strand 27.96 kb

Regulation Legend CTCF 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

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Transcript: ENSMUST00000059401

< Atg9b-201protein coding

Reverse strand 7.96 kb

ENSMUSP00000051... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam Autophagy-related protein 9 PANTHER Autophagy-related protein 9

PTHR13038:SF14

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 922

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

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