https://www.alphaknockout.com

Mouse Fam163a Knockout Project (CRISPR/Cas9)

Objective: To create a Fam163a knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fam163a gene (NCBI Reference Sequence: NM_177838 ; Ensembl: ENSMUSG00000015484 ) is located on Mouse chromosome 1. 5 exons are identified, with the ATG start codon in exon 4 and the TAA stop codon in exon 5 (Transcript: ENSMUST00000015628). Exon 4~5 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:

Exon 4 starts from about 0.2% of the coding region. Exon 4~5 covers 100.0% of the coding region. The size of effective KO region: ~1143 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 4 5

Legends Exon of mouse Fam163a 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 start codon 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 stop codon 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.

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

Sequence 12

Summary: Full Length(2000bp) | A(26.2% 524) | C(24.6% 492) | T(24.25% 485) | G(24.95% 499)

Note: The 2000 bp section upstream of start codon 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(23.35% 467) | C(23.9% 478) | T(28.3% 566) | G(24.45% 489)

Note: The 2000 bp section downstream of stop codon 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% chr1 - 156080045 156082044 2000 browser details YourSeq 199 1 1617 2000 86.5% chr6 + 86356329 86747284 390956 browser details YourSeq 175 1 327 2000 85.5% chr4 + 42039064 42039421 358 browser details YourSeq 171 29 352 2000 87.0% chr2 - 83809108 83809425 318 browser details YourSeq 170 1 348 2000 84.0% chr19 - 52626000 52626328 329 browser details YourSeq 166 1 355 2000 88.8% chr5 - 3143692 3144049 358 browser details YourSeq 166 10 349 2000 87.8% chr18 + 11953819 11954302 484 browser details YourSeq 163 1 322 2000 86.5% chr4 - 42316229 42316574 346 browser details YourSeq 158 1 333 2000 81.9% chr6 + 10280308 10280631 324 browser details YourSeq 155 19 353 2000 85.1% chr1 - 39643455 39643781 327 browser details YourSeq 155 1 354 2000 89.4% chr10 + 120315301 120315677 377 browser details YourSeq 147 1 348 2000 86.5% chr10 - 115205938 115206303 366 browser details YourSeq 147 1 224 2000 88.5% chr12 + 84585299 84585549 251 browser details YourSeq 146 1 350 2000 85.1% chr17 + 8460007 8460352 346 browser details YourSeq 145 1 292 2000 88.2% chr12 - 3095854 3096154 301 browser details YourSeq 143 1 231 2000 87.9% chr12 + 29231735 29232058 324 browser details YourSeq 140 44 348 2000 85.4% chr6 + 17180870 17181193 324 browser details YourSeq 138 1 249 2000 87.1% chr4_JH584293_random - 136349 136618 270 browser details YourSeq 138 1 249 2000 87.1% chr4_GL456350_random - 76604 76873 270 browser details YourSeq 138 1 249 2000 87.1% chr4 - 42454956 42455225 270

Note: The 2000 bp section upstream of start codon 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% chr1 - 156076900 156078899 2000 browser details YourSeq 66 1302 1424 2000 80.3% chr13 + 30725651 30725755 105 browser details YourSeq 61 1346 1425 2000 95.6% chr2 - 104344827 104344956 130 browser details YourSeq 53 1309 1387 2000 93.3% chrY - 4199940 4200053 114 browser details YourSeq 53 1354 1424 2000 93.5% chr1 - 94842600 94842671 72 browser details YourSeq 53 1347 1422 2000 83.9% chr5 + 69867199 69867273 75 browser details YourSeq 52 1309 1394 2000 78.6% chr11 + 70426034 70426096 63 browser details YourSeq 50 1343 1425 2000 80.0% chr1 + 52446766 52446834 69 browser details YourSeq 49 1347 1413 2000 93.3% chr7 + 107254227 107254299 73 browser details YourSeq 48 1347 1411 2000 80.0% chr7 - 53474320 53474376 57 browser details YourSeq 48 1310 1373 2000 89.5% chr9 + 69971398 69971461 64 browser details YourSeq 46 1352 1414 2000 88.7% chr8 + 91085823 91085882 60 browser details YourSeq 45 1308 1394 2000 71.5% chr4 + 129203047 129203096 50 browser details YourSeq 44 1343 1399 2000 94.2% chr5 + 13651945 13652009 65 browser details YourSeq 44 1314 1394 2000 79.2% chr3 + 8566349 8566407 59 browser details YourSeq 42 1307 1358 2000 83.7% chr13 - 78564708 78564756 49 browser details YourSeq 41 1368 1413 2000 85.8% chr17 - 42917793 42917834 42 browser details YourSeq 41 1316 1415 2000 68.1% chr14 - 98264932 98264980 49 browser details YourSeq 40 1309 1372 2000 95.7% chr1 - 117217178 117217328 151 browser details YourSeq 39 1310 1411 2000 67.5% chr11 - 19235215 19235258 44

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

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Gene and protein information: Fam163a family with sequence similarity 163, member A [ Mus musculus (house mouse) ] Gene ID: 329274, updated on 12-Aug-2019

Gene summary

Official Symbol Fam163a provided by MGI Official Full Name family with sequence similarity 163, member A provided by MGI Primary source MGI:MGI:3618859 See related Ensembl:ENSMUSG00000015484 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 A230106N23Rik Annotation Annotation category: suggests misassembly information Expression Orthologs Biased expression in frontal lobe adult (RPKM 2.6), kidney adult (RPKM 2.4) and 13 other tissues See more human all

Genomic context

Location: 1; 1 G3 See Fam163a in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (156075956..156205453, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (157923096..157935018, complement)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 1 transcript

Gene: Fam163a ENSMUSG00000015484

Description family with sequence similarity 163, member A [Source:MGI Symbol;Acc:MGI:3618859] Gene Synonyms A230106N23Rik Location Chromosome 1: 156,075,966-156,205,026 reverse strand. GRCm38:CM000994.2 About this gene This gene has 1 transcript (splice variant), 234 orthologues, is a member of 1 Ensembl protein family and is associated with 18 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fam163a- ENSMUST00000015628.3 3882 168aa ENSMUSP00000015628.3 Protein CCDS15390 A9ZNB6 TSL:1 201 coding Q8CAA5 GENCODE basic APPRIS P1

149.06 kb Forward strand 156.10Mb 156.15Mb 156.20Mb Genes Tor1aip2-205 >protein coding (Comprehensive set...

Tor1aip2-201 >protein coding

Contigs AC159964.5 > < AC161414.3 Genes (Comprehensive set... < Fam163a-201protein coding

Regulatory Build

156.10Mb 156.15Mb 156.20Mb Reverse strand 149.06 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

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Transcript: ENSMUST00000015628

< Fam163a-201protein coding

Reverse strand 129.06 kb

ENSMUSP00000015... Transmembrane heli... Low complexity (Seg) Pfam FAM163 PANTHER Protein FAM163A

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 168

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

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