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Mouse Gatd3a Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Gatd3a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Gatd3a gene (NCBI Reference Sequence: NM_138601.2 ; Ensembl: ENSMUSG00000053329 ) is located on Mouse chromosome 10. 7 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 7 (Transcript: ENSMUST00000001242). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Gatd3a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-77O11 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 53.01% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2464 bp, and the size of intron 6 for 3'-loxP site insertion: 678 bp. The size of effective cKO region: ~2034 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 5 6 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Gatd3a 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. 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(8534bp) | A(20.81% 1776) | C(27.53% 2349) | T(24.99% 2133) | G(26.67% 2276)

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% chr10 - 78165181 78168180 3000 browser details YourSeq 139 5 162 3000 94.4% chr5 - 31258821 31258982 162 browser details YourSeq 138 5 158 3000 94.9% chr2 - 118605907 118606060 154 browser details YourSeq 137 1 168 3000 91.1% chr15 - 36215399 36215567 169 browser details YourSeq 135 2 162 3000 92.0% chr6 + 137501456 137501616 161 browser details YourSeq 135 5 173 3000 93.1% chr1 + 97797195 97797372 178 browser details YourSeq 134 2 161 3000 91.9% chr5 - 101000717 101000876 160 browser details YourSeq 133 2 175 3000 89.4% chrX - 8048352 8048531 180 browser details YourSeq 133 2 162 3000 89.2% chr13 - 61768576 61768731 156 browser details YourSeq 133 2 162 3000 91.4% chr6 + 71380844 71381004 161 browser details YourSeq 133 1 164 3000 91.1% chr15 + 82865349 82865511 163 browser details YourSeq 132 2 173 3000 87.0% chr7 - 75722188 75722356 169 browser details YourSeq 132 2 161 3000 91.3% chr16 - 72512172 72512331 160 browser details YourSeq 132 2 160 3000 88.9% chr16 + 15689883 15690035 153 browser details YourSeq 132 2 162 3000 91.4% chr14 + 7769837 7770006 170 browser details YourSeq 132 2 161 3000 92.4% chr12 + 84953599 84953761 163 browser details YourSeq 130 9 161 3000 92.9% chr8 - 34351303 34351463 161 browser details YourSeq 130 2 161 3000 90.7% chr13 - 55050469 55050628 160 browser details YourSeq 129 2 162 3000 90.1% chr8 - 36585123 36585283 161 browser details YourSeq 129 17 162 3000 94.5% chr12 - 96532969 96533115 147

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% chr10 - 78160147 78163146 3000 browser details YourSeq 24 1675 1700 3000 96.2% chr6 + 102327721 102327746 26 browser details YourSeq 22 1320 1344 3000 95.9% chr1 - 91438974 91438999 26 browser details YourSeq 21 898 918 3000 100.0% chr3 - 141696456 141696476 21 browser details YourSeq 21 2912 2942 3000 83.9% chr14 - 121583076 121583106 31 browser details YourSeq 21 2923 2949 3000 88.9% chr1 - 66901536 66901562 27 browser details YourSeq 21 2912 2932 3000 100.0% chr12 + 80553091 80553111 21

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 protein information: Gatd3a glutamine amidotransferase like class 1 domain containing 3A [ Mus musculus (house mouse) ] Gene ID: 28295, updated on 26-Jun-2020

Gene summary

Official Symbol Gatd3a provided by MGI Official Full Name glutamine amidotransferase like class 1 domain containing 3A provided by MGI Primary source MGI:MGI:1351861 See related Ensembl:ENSMUSG00000053329 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 ES1; C21orf33; D10Jhu81e Expression Ubiquitous expression in adrenal adult (RPKM 190.1), heart adult (RPKM 107.5) and 27 other tissues See more Orthologs human all

Genomic context

Location: 10 C1; 10 39.72 cM See Gatd3a in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (78162066..78169755, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (77624812..77632513, complement)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 5 transcripts

Gene: Gatd3a ENSMUSG00000053329

Description glutamine amidotransferase like class 1 domain containing 3A [Source:MGI Symbol;Acc:MGI:1351861] Gene Synonyms D10Jhu81e Location Chromosome 10: 78,162,066-78,169,782 reverse strand. GRCm38:CM001003.2 About this gene This gene has 5 transcripts (splice variants), 256 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Gatd3a-201 ENSMUST00000001242.8 1378 266aa ENSMUSP00000001242.7 Protein coding CCDS35957 Q9D172 TSL:1 GENCODE basic APPRIS P1

Gatd3a-205 ENSMUST00000220359.1 1620 116aa ENSMUSP00000151954.1 Protein coding - A0A1W2P870 CDS 5' incomplete TSL:2

Gatd3a-203 ENSMUST00000219120.1 632 195aa ENSMUSP00000151605.1 Protein coding - A0A1W2P7B6 CDS 3' incomplete TSL:2

Gatd3a-204 ENSMUST00000219504.1 705 No protein - Retained intron - - TSL:3

Gatd3a-202 ENSMUST00000218286.1 568 No protein - Retained intron - - TSL:2

27.72 kb Forward strand

78.155Mb 78.160Mb 78.165Mb 78.170Mb 78.175Mb Contigs AC164573.5 >

Genes (Comprehensive set... < Gatd3a-201protein coding < Pwp2-201protein coding

< Gatd3a-205protein coding

< Gatd3a-203protein coding

< Gatd3a-204retained intron

< Gatd3a-202retained intron

Regulatory Build

78.155Mb 78.160Mb 78.165Mb 78.170Mb 78.175Mb Reverse strand 27.72 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

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Transcript: ENSMUST00000001242

< Gatd3a-201protein coding

Reverse strand 7.72 kb

ENSMUSP00000001... Low complexity (Seg) Superfamily Class I glutamine amidotransferase-like PANTHER PTHR10224:SF16

PTHR10224 Gene3D Class I glutamine amidotransferase-like CDD cd03133

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 266

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

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