https://www.alphaknockout.com

Mouse Puf60 Knockout Project (CRISPR/Cas9)

Objective: To create a Puf60 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Puf60 (NCBI Reference Sequence: NM_028364 ; Ensembl: ENSMUSG00000002524 ) is located on Mouse 15. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000100527). Exon 6 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 6 starts from about 21.51% of the coding region. Exon 6 covers 9.57% of the coding region. The size of effective KO region: ~162 bp. The KO region does not have any other known gene.

Page 1 of 9 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 6 12

Legends Exon of mouse Puf60 Knockout region

Page 2 of 9 https://www.alphaknockout.com

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

Forward Reverse Complement

Sequence 12

Note: The 162 bp section of Exon 6 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 162 bp section of Exon 6 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 9 https://www.alphaknockout.com

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

Sequence 12

Summary: Full Length(162bp) | A(20.99% 34) | C(27.16% 44) | T(24.07% 39) | G(27.78% 45)

Note: The 162 bp section of Exon 6 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(162bp) | A(21.6% 35) | C(27.16% 44) | T(24.07% 39) | G(27.16% 44)

Note: The 162 bp section of Exon 6 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 9 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 162 1 162 162 100.0% chr15 - 76072406 76072567 162 browser details YourSeq 21 58 79 162 100.0% chr14 - 98464161 98464183 23

Note: The 162 bp section of Exon 6 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 162 1 162 162 100.0% chr15 - 76072408 76072569 162 browser details YourSeq 24 58 88 162 72.0% chr8 - 26137402 26137426 25 browser details YourSeq 21 60 81 162 100.0% chr14 - 98464161 98464183 23

Note: The 162 bp section of Exon 6 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 https://www.alphaknockout.com

Gene and information: Puf60 poly-U binding splicing factor 60 [ Mus musculus (house mouse) ] Gene ID: 67959, updated on 15-Aug-2019

Gene summary

Official Symbol Puf60 provided by MGI Official Full Name poly-U binding splicing factor 60 provided by MGI Primary source MGI:MGI:1915209 See related Ensembl:ENSMUSG00000002524 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 SIAHBP1; 2410104I19Rik; 2810454F19Rik Expression Ubiquitous expression in ovary adult (RPKM 127.4), adrenal adult (RPKM 124.4) and 28 other tissues See more Orthologs human all

Genomic context

Location: 15; 15 D3 See Puf60 in Genome Data Viewer Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (76070182..76082537, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (75900612..75911376, complement)

Chromosome 15 - NC_000081.6

Page 6 of 9 https://www.alphaknockout.com

Transcript information: This gene has 11 transcripts

Gene: Puf60 ENSMUSG00000002524

Description poly-U binding splicing factor 60 [Source:MGI Symbol;Acc:MGI:1915209] Gene Synonyms 2410104I19Rik, 2810454F19Rik Location Chromosome 15: 76,070,182-76,080,924 reverse strand. GRCm38:CM001008.2 About this gene This gene has 11 transcripts (splice variants), 211 orthologues, 23 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Puf60- ENSMUST00000100527.12 1881 564aa ENSMUSP00000098096.4 Protein coding CCDS37112 Q3UEB3 TSL:1 202 GENCODE basic APPRIS P4

Puf60- ENSMUST00000002599.10 1821 547aa ENSMUSP00000002599.9 Protein coding CCDS37111 Q3UEB3 TSL:1 201 GENCODE basic APPRIS ALT2

Puf60- ENSMUST00000227316.1 1825 140aa ENSMUSP00000154177.1 Nonsense mediated - A0A2I3BQR9 CDS 5' 204 decay incomplete

Puf60- ENSMUST00000230924.1 652 137aa ENSMUSP00000155707.1 Nonsense mediated - A0A2R8VI42 - 210 decay

Puf60- ENSMUST00000228459.1 1008 No - Retained intron - - - 205 protein

Puf60- ENSMUST00000229474.1 831 No - Retained intron - - - 207 protein

Puf60- ENSMUST00000230968.1 769 No - Retained intron - - - 211 protein

Puf60- ENSMUST00000227251.1 429 No - Retained intron - - - 203 protein

Puf60- ENSMUST00000229030.1 349 No - Retained intron - - - 206 protein

Puf60- ENSMUST00000230143.1 728 No - lncRNA - - - 209 protein

Puf60- ENSMUST00000229496.1 586 No - lncRNA - - - 208 protein

Page 7 of 9 https://www.alphaknockout.com

30.74 kb Forward strand 76.07Mb 76.08Mb 76.09Mb Gm48952-201 >lncRNA (Comprehensive set...

Contigs < AC116487.14 Genes (Comprehensive set... < Scrib-203protein coding < Puf60-202protein coding < Nrbp2-205nonsense mediated decay

< Scrib-202protein coding < Puf60-201protein coding < Nrbp2-203nonsense mediated decay

< Scrib-201protein coding < Puf60-204nonsense mediated decay < Nrbp2-209nonsense mediated decay

< Scrib-204lncRNA < Mir6952-201miRNA < Puf60-203retained intron < Puf60-206retained intron < Nrbp2-208protein coding

< Puf60-207retained intron < Puf60-205retained intron < Nrbp2-207retained intron

< Puf60-210nonsense mediated decay < Nrbp2-201protein coding

< Puf60-211retained intron < Nrbp2-204retained intron

< Puf60-208lncRNA < Nrbp2-206retained intron

< Puf60-209lncRNA < Nrbp2-202lncRNA

< Nrbp2-210lncRNA

Regulatory Build

76.07Mb 76.08Mb 76.09Mb Reverse strand 30.74 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

Page 8 of 9 https://www.alphaknockout.com

Transcript: ENSMUST00000100527

< Puf60-202protein coding

Reverse strand 10.73 kb

ENSMUSP00000098... MobiDB lite Low complexity (Seg) TIGRFAM Poly-U binding splicing factor, half-pint Superfamily RNA-binding domain superfamily SMART RNA recognition motif domain, eukaryote

RNA recognition motif domain Pfam RNA recognition motif domain PROSITE profiles RNA recognition motif domain PANTHER PTHR47330 Gene3D Nucleotide-binding alpha-beta plait domain superfamily CDD PUF60, RNA recognition motif 2 PUF60, RNA recognition motif 3

PUF60, RNA recognition motif 1

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

Variant Legend synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 564

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

Page 9 of 9