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Mouse Arpc3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Arpc3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Arpc3 gene (NCBI Reference Sequence: NM_019824 ; Ensembl: ENSMUSG00000029465 ) is located on Mouse chromosome 5. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000102525). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Arpc3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-393K21 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 transposon-induced allele develop only to the blastocyst stage and show defects in trophoblast outgrowth and in the dynamics of actin accumulation. Mice heterozygous for the same transposon-induced allele and a knock-out allele showimpaired trophoblast outgrowth activity.

Exon 3 starts from about 20.04% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 4730 bp, and the size of intron 3 for 3'-loxP site insertion: 1635 bp. The size of effective cKO region: ~577 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 3 4 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Arpc3 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(7077bp) | A(23.58% 1669) | C(23.71% 1678) | T(27.0% 1911) | G(25.7% 1819)

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% chr5 + 122398398 122401397 3000 browser details YourSeq 144 40 218 3000 91.5% chr11 + 79171456 79171652 197 browser details YourSeq 136 71 652 3000 79.0% chr4 + 118299152 118299375 224 browser details YourSeq 134 43 238 3000 85.8% chr15 - 98712020 98712226 207 browser details YourSeq 133 39 230 3000 85.8% chr7 - 138600822 138601021 200 browser details YourSeq 131 44 228 3000 87.1% chr4 - 116711504 116711688 185 browser details YourSeq 130 42 711 3000 79.3% chr12 - 55445431 55445689 259 browser details YourSeq 130 41 228 3000 87.9% chr11 - 109133556 109133748 193 browser details YourSeq 129 53 230 3000 87.8% chr5 + 121477317 121477499 183 browser details YourSeq 128 41 218 3000 86.6% chr12 + 87209252 87209432 181 browser details YourSeq 125 40 709 3000 74.2% chr9 + 44610789 44611038 250 browser details YourSeq 125 42 230 3000 85.8% chr3 + 116336129 116336331 203 browser details YourSeq 123 40 213 3000 85.7% chr8 - 36178907 36179081 175 browser details YourSeq 123 37 230 3000 82.4% chr7 + 78873261 78873446 186 browser details YourSeq 123 47 218 3000 87.0% chr10 + 61592840 61593015 176 browser details YourSeq 122 45 709 3000 74.0% chr6 + 54837302 54837550 249 browser details YourSeq 121 41 228 3000 90.2% chr2 - 60266427 60266617 191 browser details YourSeq 119 50 218 3000 89.5% chr15 + 76641623 76641793 171 browser details YourSeq 118 41 218 3000 83.8% chr3 - 97979892 97980072 181 browser details YourSeq 118 40 208 3000 85.3% chr2 - 25008559 25008728 170

Note: The 3000 bp section upstream of Exon 3 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 + 122401975 122404974 3000 browser details YourSeq 151 4 1894 3000 83.4% chr2 - 25032508 25300909 268402 browser details YourSeq 123 22 1818 3000 90.2% chr15 + 72992253 73237352 245100 browser details YourSeq 86 1748 1887 3000 80.8% chr4 - 57879639 57879778 140 browser details YourSeq 79 1746 1880 3000 79.7% chr15 + 100752427 100752561 135 browser details YourSeq 79 1748 1891 3000 82.2% chr11 + 106356538 106357011 474 browser details YourSeq 77 1752 1873 3000 87.4% chr10 - 28202959 28203082 124 browser details YourSeq 77 1748 1876 3000 79.9% chr11 + 115593335 115593463 129 browser details YourSeq 76 1746 1875 3000 80.2% chr1 + 134302010 134302140 131 browser details YourSeq 73 11 104 3000 84.6% chr17 - 87497206 87497289 84 browser details YourSeq 72 22 155 3000 79.1% chr2 + 177721439 177721530 92 browser details YourSeq 71 22 104 3000 87.4% chr4 - 123365580 123365658 79 browser details YourSeq 71 23 110 3000 92.8% chr4 + 38263679 38263766 88 browser details YourSeq 69 22 105 3000 87.9% chr1 - 131144248 131144329 82 browser details YourSeq 69 22 110 3000 88.8% chr19 + 56410211 56410299 89 browser details YourSeq 68 2 91 3000 87.8% chr9 - 37584231 37584320 90 browser details YourSeq 68 20 103 3000 89.1% chr8 - 78708604 78708686 83 browser details YourSeq 68 22 104 3000 90.0% chr6 - 135636164 135636245 82 browser details YourSeq 68 22 110 3000 83.6% chr18 - 26907144 26907223 80 browser details YourSeq 68 22 95 3000 96.0% chr5 + 37535679 37535752 74

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Arpc3 actin related protein 2/3 complex, subunit 3 [ Mus musculus (house mouse) ] Gene ID: 56378, updated on 12-Aug-2019

Gene summary

Official Symbol Arpc3 provided by MGI Official Full Name actin related protein 2/3 complex, subunit 3 provided by MGI Primary source MGI:MGI:1928375 See related Ensembl:ENSMUSG00000029465 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 p21-Ar; p21Arc; p21-ARC; 1110006A04Rik Expression Ubiquitous expression in placenta adult (RPKM 128.4), large intestine adult (RPKM 106.1) and 28 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 F See Arpc3 in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (122391878..122406181)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (122841937..122856187)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 8 transcripts

Gene: Arpc3 ENSMUSG00000029465

Description actin related protein 2/3 complex, subunit 3 [Source:MGI Symbol;Acc:MGI:1928375] Gene Synonyms 1110006A04Rik, Arp2/3 complex subunit p21-Arc, p21-Ar Location Chromosome 5: 122,391,878-122,414,184 forward strand. GRCm38:CM000998.2 About this gene This gene has 8 transcripts (splice variants), 209 orthologues, is a member of 1 Ensembl protein family and is associated with 13 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Arpc3-202 ENSMUST00000102525.10 899 178aa ENSMUSP00000099584.4 Protein coding CCDS19648 Q9JM76 TSL:1 GENCODE basic APPRIS P1

Arpc3-204 ENSMUST00000111716.7 838 161aa ENSMUSP00000107345.1 Protein coding - D3Z2F7 TSL:2 GENCODE basic

Arpc3-201 ENSMUST00000031421.11 736 170aa ENSMUSP00000031421.5 Protein coding - H7BWZ3 TSL:3 GENCODE basic

Arpc3-203 ENSMUST00000111713.1 708 163aa ENSMUSP00000107342.1 Protein coding - D3Z2F8 TSL:3 GENCODE basic

Arpc3-208 ENSMUST00000196969.4 599 142aa ENSMUSP00000143210.1 Protein coding - A0A0G2JFK7 CDS 5' incomplete TSL:3

Arpc3-207 ENSMUST00000148913.1 856 No protein - Retained intron - - TSL:2

Arpc3-205 ENSMUST00000126247.1 672 No protein - Retained intron - - TSL:5

Arpc3-206 ENSMUST00000141395.1 409 No protein - Retained intron - - TSL:2

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42.31 kb Forward strand 122.39Mb 122.40Mb 122.41Mb 122.42Mb Genes (Comprehensive set... Gpn3-203 >nonsense mediated decay Arpc3-202 >protein coding Gm42829-201 >lncRNA Anapc7-203 >protein coding

Gpn3-201 >protein coding Arpc3-207 >retained intron Arpc3-205 >retained intron Anapc7-201 >protein coding

Gpn3-202 >nonsense mediated decay Arpc3-206 >retained intron Arpc3-204 >protein coding Anapc7-202 >protein coding

Gpn3-204 >nonsense mediated decay Arpc3-201 >protein coding

Gpn3-205 >protein coding Arpc3-208 >protein coding

Arpc3-203 >protein coding

Contigs < AC113285.9 Regulatory Build

122.39Mb 122.40Mb 122.41Mb 122.42Mb Reverse strand 42.31 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000102525

14.30 kb Forward strand

Arpc3-202 >protein coding

ENSMUSP00000099... Superfamily Actin-related protein 2/3 complex subunit 3 superfamily

Pfam Actin-related protein 2/3 complex subunit 3

PIRSF Actin-related protein 2/3 complex subunit 3

PANTHER Actin-related protein 2/3 complex subunit 3

PTHR12391:SF1 Gene3D Actin-related protein 2/3 complex subunit 3 superfamily

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

Variant Legend

splice region variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 178

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

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