https://www.alphaknockout.com

Mouse Actr2 Knockout Project (CRISPR/Cas9)

Objective: To create a Actr2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Actr2 gene (NCBI Reference Sequence: NM_146243 ; Ensembl: ENSMUSG00000020152 ) is located on Mouse chromosome 11. 9 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000000137). Exon 2~4 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice heterozygous for this mutation exhibit modifies lethality associated with F5 null Tfpi heterozygous mice.

Exon 2 starts from about 4.15% of the coding region. Exon 2~4 covers 33.84% of the coding region. The size of effective KO region: ~9639 bp. The KO 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 9

Legends Exon of mouse Actr2 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

Overview of the Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 4 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(27.7% 554) | C(21.35% 427) | T(29.9% 598) | G(21.05% 421)

Note: The 2000 bp section upstream of Exon 2 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(26.1% 522) | C(21.7% 434) | T(30.2% 604) | G(22.0% 440)

Note: The 2000 bp section downstream of Exon 4 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 2000 1 2000 2000 100.0% chr11 - 20100934 20102933 2000 browser details YourSeq 413 1 456 2000 95.4% chr5 + 8753232 8753689 458 browser details YourSeq 410 1 456 2000 95.4% chr5 + 68084206 68084663 458 browser details YourSeq 410 1 457 2000 95.0% chr1 + 191949285 191949743 459 browser details YourSeq 409 1 456 2000 95.0% chrX + 105467743 105468200 458 browser details YourSeq 409 1 953 2000 89.6% chr6 + 9943003 9943485 483 browser details YourSeq 406 1 457 2000 94.6% chr11 + 19841392 19841850 459 browser details YourSeq 405 1 456 2000 95.0% chr19 - 46677965 46678420 456 browser details YourSeq 401 1 456 2000 94.1% chrX + 95761170 95761627 458 browser details YourSeq 401 1 953 2000 88.8% chr7 + 42138131 42138613 483 browser details YourSeq 401 1 457 2000 95.7% chr19 + 25256252 25256716 465 browser details YourSeq 400 1 464 2000 93.5% chr9 + 49352730 49353195 466 browser details YourSeq 399 1 456 2000 93.9% chr8 - 126243494 126243951 458 browser details YourSeq 398 1 462 2000 93.9% chr9 - 18448933 18449387 455 browser details YourSeq 398 1 457 2000 93.7% chr9 + 82006759 82007217 459 browser details YourSeq 398 1 457 2000 93.7% chr8 + 122165864 122166322 459 browser details YourSeq 397 2 457 2000 93.7% chrX + 112335181 112335638 458 browser details YourSeq 397 1 456 2000 94.6% chr6 + 100034059 100034514 456 browser details YourSeq 397 1 456 2000 93.7% chr6 + 96482749 96483205 457 browser details YourSeq 397 1 456 2000 93.7% chr5 + 24222167 24222624 458

Note: The 2000 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 2000 1 2000 2000 100.0% chr11 - 20089295 20091294 2000 browser details YourSeq 200 63 272 2000 98.6% chr1 + 78415618 78416164 547 browser details YourSeq 198 63 353 2000 96.3% chr6 - 51074184 51074493 310 browser details YourSeq 197 65 264 2000 99.5% chr10 - 127638508 127638726 219 browser details YourSeq 196 73 289 2000 97.2% chr3 + 96605991 96606651 661 browser details YourSeq 195 63 263 2000 99.0% chr11 - 17975351 17975552 202 browser details YourSeq 195 56 274 2000 98.1% chrX + 21053290 21053509 220 browser details YourSeq 195 62 267 2000 96.6% chr2 + 127272066 127272270 205 browser details YourSeq 195 81 419 2000 94.0% chr15 + 8085897 8086233 337 browser details YourSeq 194 60 263 2000 99.0% chr17 - 46727555 46727777 223 browser details YourSeq 194 66 262 2000 98.0% chr11 - 107441377 107441571 195 browser details YourSeq 193 68 265 2000 99.0% chr8 - 125661879 125662078 200 browser details YourSeq 193 63 263 2000 98.5% chr4 - 43456319 43456520 202 browser details YourSeq 193 63 261 2000 99.0% chr14 - 34340428 34340627 200 browser details YourSeq 193 63 277 2000 97.6% chr12 + 8768322 8768545 224 browser details YourSeq 193 63 261 2000 99.0% chr1 + 38072195 38072394 200 browser details YourSeq 192 60 261 2000 98.1% chrX - 66405162 66405375 214 browser details YourSeq 192 64 262 2000 98.5% chr2 - 153480836 153481038 203 browser details YourSeq 191 63 261 2000 98.5% chr17 - 24098897 24099096 200 browser details YourSeq 191 63 262 2000 98.0% chr9 + 56988984 56989186 203

Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Actr2 ARP2 actin-related protein 2 [ Mus musculus (house mouse) ] Gene ID: 66713, updated on 27-Aug-2019

Gene summary

Official Symbol Actr2 provided by MGI Official Full Name ARP2 actin-related protein 2 provided by MGI Primary source MGI:MGI:1913963 See related Ensembl:ENSMUSG00000020152 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 Arp2; AA409782; D6Ertd746e; 4921510D23Rik Expression Ubiquitous expression in placenta adult (RPKM 51.3), CNS E18 (RPKM 43.4) and 25 other tissues See more Orthologs human all

Genomic context

Location: 11 A3.1; 11 12.88 cM See Actr2 in Genome Data Viewer Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (20062304..20112951, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (19962307..20012954, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 3 transcripts

Gene: Actr2 ENSMUSG00000020152

Description ARP2 actin-related protein 2 [Source:MGI Symbol;Acc:MGI:1913963] Gene Synonyms 4921510D23Rik, Arp2, D6Ertd746e Location Chromosome 11: 20,062,304-20,112,913 reverse strand. GRCm38:CM001004.2 About this gene This gene has 3 transcripts (splice variants), 245 orthologues, 27 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Actr2-201 ENSMUST00000000137.7 3631 394aa ENSMUSP00000000137.7 Protein coding CCDS24455 P61161 Q5SW83 TSL:1 GENCODE basic APPRIS P1

Actr2-203 ENSMUST00000134779.1 452 No protein - Retained intron - - TSL:2

Actr2-202 ENSMUST00000132022.1 2087 No protein - lncRNA - - TSL:1

70.61 kb Forward strand 20.06Mb 20.08Mb 20.10Mb 20.12Mb Contigs AL606522.6 > Genes < Actr2-201protein coding (Comprehensive set...

< Actr2-202lncRNA

< Actr2-203retained intron

Regulatory Build

20.06Mb 20.08Mb 20.10Mb 20.12Mb Reverse strand 70.61 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000000137

< Actr2-201protein coding

Reverse strand 50.61 kb

ENSMUSP00000000... Low complexity (Seg) Superfamily SSF53067 SMART Actin family Prints Actin family Pfam Actin family PROSITE patterns Actin/actin-like conserved site PANTHER Actin family

Actin-related protein 2 Gene3D 3.30.420.40

3.90.640.10 CDD cd00012

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 394

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

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