https://www.alphaknockout.com

Mouse Fam19a5 Knockout Project (CRISPR/Cas9)

Objective: To create a Fam19a5 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fam19a5 (NCBI Reference Sequence: NM_001252310 ; Ensembl: ENSMUSG00000054863 ) is located on Mouse 15. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000230414). Exon 2 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 2 starts from about 28.54% of the coding region. Exon 2 covers 37.88% of the coding region. The size of effective KO region: ~150 bp. The KO region does not have any other known gene.

Page 1 of 8 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 4

Legends Exon of mouse Fam19a5 Knockout region

Page 2 of 8 https://www.alphaknockout.com

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

Forward Reverse Complement

Sequence 12

Note: The 150 bp section of Exon 2 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 150 bp section of Exon 2 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.

Page 3 of 8 https://www.alphaknockout.com

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

Sequence 12

Summary: Full Length(150bp) | A(22.0% 33) | C(30.0% 45) | T(13.33% 20) | G(34.67% 52)

Note: The 150 bp section 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(150bp) | A(22.0% 33) | C(30.0% 45) | T(13.33% 20) | G(34.67% 52)

Note: The 150 bp section 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.

Page 4 of 8 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 150 1 150 150 100.0% chr15 + 87681482 87681631 150

Note: The 150 bp section 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 150 1 150 150 100.0% chr15 + 87681480 87681629 150 browser details YourSeq 21 17 40 150 95.9% chr11 + 53708591 53708615 25

Note: The 150 bp section of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 8 https://www.alphaknockout.com

Gene and information: Tafa5 TAFA chemokine like family member 5 [ Mus musculus (house mouse) ] Gene ID: 106014, updated on 24-Oct-2019

Gene summary

Official Symbol Tafa5 provided by MGI Official Full Name TAFA chemokine like family member 5 provided by MGI Primary source MGI:MGI:2146182 See related Ensembl:ENSMUSG00000054863 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Tafa-5; Tara-5; Fam19a5 Summary This gene is a member of the TAFA family which is composed of five highly homologous that encode small secreted Expression . These proteins contain conserved cysteine residues at fixed positions, and are distantly related to MIP-1alpha, a member of the CC-chemokine family. The TAFA proteins are predominantly expressed in specific regions of the brain, and are postulated to function as brain-specific chemokines or neurokines, that act as regulators of immune and nervous cells. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Nov 2011] Orthologs Biased expression in frontal lobe adult (RPKM 18.4), cerebellum adult (RPKM 17.8) and 14 other tissues See more human all

Genomic context

Location: 15; 15 E3 See Tafa5 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (87544219..87759364)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (87455660..87589794)

Chromosome 15 - NC_000081.6

Page 6 of 8 https://www.alphaknockout.com

Transcript information: This gene has 2 transcripts

Gene: Tafa5 ENSMUSG00000054863

Description TAFA chemokine like family member 5 [Source:MGI Symbol;Acc:MGI:2146182] Gene Synonyms Fam19a5, Tara-5 Location Chromosome 15: 87,544,299-87,759,364 forward strand. GRCm38:CM001008.2 About this gene This gene has 2 transcripts (splice variants), 225 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tafa5-201 ENSMUST00000068088.7 2603 125aa ENSMUSP00000064808.6 Protein coding CCDS49693 Q91WE9 TSL:1 GENCODE basic

Tafa5-202 ENSMUST00000230414.1 2649 132aa ENSMUSP00000155322.1 Protein coding - Q91WE9 GENCODE basic APPRIS P1

235.07 kb Forward strand 87.55Mb 87.60Mb 87.65Mb 87.70Mb 87.75Mb Genes (Comprehensive set... Tafa5-202 >protein coding

Tafa5-201 >protein coding

Contigs AC124381.4 > < AC124449.3 AC123925.4 > Regulatory Build

87.55Mb 87.60Mb 87.65Mb 87.70Mb 87.75Mb Reverse strand 235.07 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000230414

215.06 kb Forward strand

Tafa5-202 >protein coding

ENSMUSP00000155... Transmembrane heli... Pfam Chemokine-like protein TAFA

PANTHER PTHR31878:SF0

Chemokine-like protein TAFA-5

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

Variant Legend

missense variant synonymous variant

Scale bar 0 20 40 60 80 100 132

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

Page 8 of 8