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Mouse Arpc5l Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Arpc5l conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Arpc5l gene (NCBI Reference Sequence: NM_028809 ; Ensembl: ENSMUSG00000026755 ) is located on Mouse chromosome 2. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000112862). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Arpc5l gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-82N14 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 2 starts from about 32.68% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 4770 bp, and the size of intron 3 for 3'-loxP site insertion: 1158 bp. The size of effective cKO region: ~1285 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 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Arpc5l 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. 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(7785bp) | A(26.22% 2041) | C(20.08% 1563) | T(29.44% 2292) | G(24.26% 1889)

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% chr2 + 39009866 39012865 3000 browser details YourSeq 195 202 423 3000 93.6% chr6 + 91061683 91061903 221 browser details YourSeq 190 216 407 3000 99.5% chr5 - 65425625 65425816 192 browser details YourSeq 188 216 407 3000 98.0% chr11 + 52356558 52356748 191 browser details YourSeq 187 132 407 3000 95.2% chr9 + 99320282 99320675 394 browser details YourSeq 186 200 420 3000 94.0% chr12 - 84737954 84738182 229 browser details YourSeq 185 129 407 3000 93.4% chr2 - 33392436 33392766 331 browser details YourSeq 185 208 435 3000 93.1% chr8 + 88210250 88210507 258 browser details YourSeq 185 202 423 3000 94.8% chr7 + 130180020 130180261 242 browser details YourSeq 182 222 407 3000 99.0% chr17 - 29027903 29028088 186 browser details YourSeq 182 204 426 3000 89.9% chr12 - 31096201 31096410 210 browser details YourSeq 181 226 407 3000 100.0% chr8 - 110856297 110856480 184 browser details YourSeq 181 208 415 3000 93.8% chr12 + 80561191 80561401 211 browser details YourSeq 181 85 407 3000 87.6% chr11 + 96359987 96360261 275 browser details YourSeq 180 208 407 3000 95.5% chr14 + 19792190 19792390 201 browser details YourSeq 179 206 409 3000 94.1% chr9 - 83733116 83733321 206 browser details YourSeq 179 205 409 3000 94.6% chr7 - 19071986 19072196 211 browser details YourSeq 179 206 407 3000 95.0% chr16 - 22758306 22758507 202 browser details YourSeq 179 208 410 3000 94.6% chr14 - 49984405 49984608 204 browser details YourSeq 179 210 410 3000 95.5% chr11 - 116691819 116692039 221

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% chr2 + 39014151 39017150 3000 browser details YourSeq 90 2113 2494 3000 75.8% chr8 - 33590566 33590692 127 browser details YourSeq 85 2105 2494 3000 74.6% chr15 + 103267287 103267421 135 browser details YourSeq 85 2108 2261 3000 92.0% chr10 + 128253286 128253700 415 browser details YourSeq 81 2113 2494 3000 75.3% chr6 - 31466295 31466421 127 browser details YourSeq 81 2100 2494 3000 77.0% chr10 - 13351701 13351850 150 browser details YourSeq 81 2114 2494 3000 89.6% chr2 + 84657351 84657736 386 browser details YourSeq 80 2113 2282 3000 80.5% chr1 - 159648896 159649046 151 browser details YourSeq 79 2105 2212 3000 87.1% chr16 - 4440339 4440439 101 browser details YourSeq 79 2104 2262 3000 90.5% chr10 - 26320219 26320379 161 browser details YourSeq 78 2114 2494 3000 75.0% chr19 + 37463126 37463251 126 browser details YourSeq 77 2113 2262 3000 82.3% chrX + 40969271 40969412 142 browser details YourSeq 74 2113 2494 3000 73.3% chr5 - 42741564 42741690 127 browser details YourSeq 74 2113 2201 3000 92.1% chr14 - 52113908 52113996 89 browser details YourSeq 74 2113 2260 3000 90.5% chr1 - 150520983 150521162 180 browser details YourSeq 74 2110 2209 3000 81.4% chr6 + 24363406 24363497 92 browser details YourSeq 74 2126 2283 3000 95.1% chr13 + 11145690 11145847 158 browser details YourSeq 73 2113 2209 3000 83.2% chr13 + 69220838 69220927 90 browser details YourSeq 72 2113 2494 3000 72.8% chr1 - 84992000 84992126 127 browser details YourSeq 72 2113 2281 3000 79.1% chr7 + 61826571 61826716 146

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: Arpc5l actin related protein 2/3 complex, subunit 5-like [ Mus musculus (house mouse) ] Gene ID: 74192, updated on 12-Aug-2019

Gene summary

Official Symbol Arpc5l provided by MGI Official Full Name actin related protein 2/3 complex, subunit 5-like provided by MGI Primary source MGI:MGI:1921442 See related Ensembl:ENSMUSG00000026755 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as ARC16-2; AI852867; AW555592; AW742746; 2010015J01Rik Expression Ubiquitous expression in spleen adult (RPKM 20.9), testis adult (RPKM 20.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 B See Arpc5l in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (39008066..39015878)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (38863659..38871392)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 5 transcripts

Gene: Arpc5l ENSMUSG00000026755

Description actin related protein 2/3 complex, subunit 5-like [Source:MGI Symbol;Acc:MGI:1921442] Gene Synonyms 2010015J01Rik, ARC16-2 Location Chromosome 2: 39,005,348-39,015,877 forward strand. GRCm38:CM000995.2 About this gene This gene has 5 transcripts (splice variants), 164 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Arpc5l- ENSMUST00000112862.6 1339 153aa ENSMUSP00000108483.2 Protein coding CCDS38120 Q9D898 TSL:1 202 GENCODE basic APPRIS P1

Arpc5l- ENSMUST00000090993.7 752 65aa ENSMUSP00000088516.6 Nonsense mediated - A0A0R3P9C9 TSL:2 201 decay

Arpc5l- ENSMUST00000135049.1 4167 No - lncRNA - - TSL:1 203 protein

Arpc5l- ENSMUST00000141467.7 579 No - lncRNA - - TSL:3 204 protein

Arpc5l- ENSMUST00000204825.2 361 No - lncRNA - - TSL:2 205 protein

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30.53 kb Forward strand 39.00Mb 39.01Mb 39.02Mb Genes (Comprehensive set... Wdr38-201 >nonsense mediated decay Arpc5l-202 >protein coding

Wdr38-202 >protein coding Arpc5l-204 >lncRNA

Wdr38-203 >lncRNA Arpc5l-205 >lncRNA

Arpc5l-201 >nonsense mediated decay

Arpc5l-203 >lncRNA

Contigs AL844588.17 > Genes < Gm13496-201lncRNA < Rpl35-201protein coding < Golga1-201protein coding (Comprehensive set...

< Rpl35-203lncRNA < Golga1-202protein coding

< Rpl35-202lncRNA < Golga1-208nonsense mediated decay

< Golga1-203lncRNA < Golga1-204retained intron

< Golga1-210nonsense mediated decay

< Golga1-211retained intron

< Golga1-209nonsense mediated decay

< Golga1-207retained intron

Regulatory Build

39.00Mb 39.01Mb 39.02Mb Reverse strand 30.53 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000112862

7.80 kb Forward strand

Arpc5l-202 >protein coding

ENSMUSP00000108... Low complexity (Seg) Superfamily Actin-related protein 2/3 complex subunit 5 superfamily Pfam Actin-related protein 2/3 complex subunit 5 PIRSF Actin-related protein 2/3 complex subunit 5 PANTHER Actin-related protein 2/3 complex subunit 5-like protein

Actin-related protein 2/3 complex subunit 5 Gene3D Actin-related protein 2/3 complex subunit 5 superfamily

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

Variant Legend inframe insertion missense variant

Scale bar 0 20 40 60 80 100 120 153

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

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