http://beta.alphaknockout.cyagen.net

Mouse Ppp1r13l Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ppp1r13l conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ppp1r13l gene (NCBI Reference Sequence: NM_001010836 ; Ensembl: ENSMUSG00000040734 ) is located on Mouse chromosome 7. 13 exons are identified , with the ATG start codon in exon 2 and the TAG stop codon in exon 13 (Transcript: ENSMUST00000047621). Exon 4~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ppp1r13l gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-258L20 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: Homozygotes for spontaneous mutations in this gene exhibit cardiovascular defects leading to cardiomyopathy, open eyelids at birth, and coat abnormalities. One allele also shows postnatal lethality dependent on strain background and decreased weight, while another shows impaired fertility.

The knockout of Exon 4~7 will result in frameshift of the gene, and covers 46.28% of the coding region. The size of intron 3 for 5'-loxP site insertion: 934 bp, and the size of intron 7 for 3'-loxP site insertion: 1277 bp.

The size of effective cKO region: ~2796 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level.

Page 1 of 8 http://beta.alphaknockout.cyagen.net

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 5 6 7 8 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ppp1r13l Homology arm cKO region loxP site

Page 2 of 8 http://beta.alphaknockout.cyagen.net

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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8853bp) | A(23.42% 2073) | C(26.3% 2328) | G(25.0% 2213) | T(25.29% 2239)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

Page 3 of 8 http://beta.alphaknockout.cyagen.net

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr7 + 19366545 19369544 3000 browser details YourSeq 126 1497 1649 3000 95.7% chr5 - 144625047 144625408 362 browser details YourSeq 125 1507 1649 3000 94.4% chr7 - 35105896 35106094 199 browser details YourSeq 124 1507 1649 3000 93.7% chr7 + 30304321 30304463 143 browser details YourSeq 124 1507 1649 3000 92.2% chr5 + 138926546 138926687 142 browser details YourSeq 123 1507 1649 3000 93.7% chr5 + 144049366 144049562 197 browser details YourSeq 122 1507 1649 3000 90.0% chr5 + 141075029 141075168 140 browser details YourSeq 122 1514 1649 3000 95.6% chr5 + 139680219 139680406 188 browser details YourSeq 122 1507 1649 3000 93.0% chr5 + 141395704 141395846 143 browser details YourSeq 121 1521 1649 3000 96.9% chr7 - 36560202 36560330 129 browser details YourSeq 120 1521 1649 3000 96.9% chr7 - 36024909 36025038 130 browser details YourSeq 120 1521 1649 3000 96.9% chr7 - 35765313 35765442 130 browser details YourSeq 120 1507 1649 3000 92.4% chr7 - 16194577 16194720 144 browser details YourSeq 120 1507 1649 3000 92.3% chr5 - 139494363 139494505 143 browser details YourSeq 120 1521 1649 3000 96.9% chr5 - 140550309 140550438 130 browser details YourSeq 120 1521 1649 3000 96.9% chr5 - 140011958 140012087 130 browser details YourSeq 120 1521 1649 3000 96.9% chr5 - 140011905 140012034 130 browser details YourSeq 120 1521 1649 3000 96.9% chr7 + 34569326 34569455 130 browser details YourSeq 120 1521 1649 3000 96.9% chr7 + 18946078 18946207 130 browser details YourSeq 119 1521 1649 3000 96.9% chr5 - 140979615 141002193 22579

Note: The 3000 bp section upstream of Exon 4 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% chr7 + 19371898 19374897 3000 browser details YourSeq 164 2379 2973 3000 87.3% chr10 + 83776429 83777074 646 browser details YourSeq 150 2379 3000 3000 81.6% chr2 - 69081845 69082153 309 browser details YourSeq 141 2548 2961 3000 88.9% chr1 - 93084515 93085008 494 browser details YourSeq 139 2797 2996 3000 91.3% chr7 - 29416680 29416996 317 browser details YourSeq 138 2817 3000 3000 86.2% chr9 + 53934552 53934733 182 browser details YourSeq 135 2416 2979 3000 81.0% chr2 - 113706196 113706447 252 browser details YourSeq 134 2379 2954 3000 77.1% chr6 - 32505371 32505702 332 browser details YourSeq 131 2387 2977 3000 80.8% chr9 + 71697691 71697994 304 browser details YourSeq 131 2817 3000 3000 85.8% chr7 + 35957704 35957887 184 browser details YourSeq 130 2826 2999 3000 88.5% chr7 - 144804134 144804306 173 browser details YourSeq 126 2819 3000 3000 86.6% chr9 - 107721005 107721184 180 browser details YourSeq 123 2820 3000 3000 86.4% chr16 - 28713107 28713288 182 browser details YourSeq 123 2820 2974 3000 88.4% chr4 + 106071743 106071896 154 browser details YourSeq 122 2453 2999 3000 78.8% chr10 + 116951535 116951906 372 browser details YourSeq 119 2820 2974 3000 87.1% chr2 - 105449781 105449934 154 browser details YourSeq 118 2601 2996 3000 85.1% chr7 + 116995960 116996541 582 browser details YourSeq 117 2817 3000 3000 86.7% chr6 + 40761461 40761642 182 browser details YourSeq 117 2848 3000 3000 85.3% chr17 + 4436200 4436348 149 browser details YourSeq 117 2842 3000 3000 84.8% chr14 + 61862766 61862922 157

Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 http://beta.alphaknockout.cyagen.net Gene and protein information: Ppp1r13l protein phosphatase 1, regulatory subunit 13 like [ Mus musculus (house mouse) ] Gene ID: 333654, updated on 21-Sep-2019

Gene summary

Official Symbol Ppp1r13l provided by MGI Official Full Name protein phosphatase 1, regulatory subunit 13 like provided by MGI Primary source MGI:MGI:3525053 See related Ensembl:ENSMUSG00000040734 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 wa3; IASPP; Nkip1; AV260376 Expression Broad expression in testis adult (RPKM 16.3), stomach adult (RPKM 11.1) and 23 other tissues See more Orthologs human all

Genomic context

Location: 7 A3; 7 9.62 cM See Ppp1r13l in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (19359724..19378540)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (19946565..19963882)

Chromosome 7 - NC_000073.6

Page 5 of 8 http://beta.alphaknockout.cyagen.net

Transcript information: This gene has 5 transcripts

Gene: Ppp1r13l ENSMUSG00000040734

Description protein phosphatase 1, regulatory subunit 13 like [Source:MGI Symbol;Acc:MGI:3525053] Gene Synonyms IASPP, NFkB interacting protein 1, wa3 Location Chromosome 7: 19,359,749-19,378,533 forward strand. GRCm38:CM001000.2 About this gene This gene has 5 transcripts (splice variants), 161 orthologues, is a member of 1 Ensembl protein family and is associated with 55 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ppp1r13l- ENSMUST00000047621.13 3130 824aa ENSMUSP00000047839.7 Protein CCDS20899 Q3TCU2 TSL:1 201 coding Q5I1X5 GENCODE basic APPRIS P1

Ppp1r13l- ENSMUST00000127785.7 841 112aa ENSMUSP00000116351.1 Protein - D3Z5Q5 CDS 3' 202 coding incomplete TSL:5

Ppp1r13l- ENSMUST00000140836.7 720 114aa ENSMUSP00000114443.1 Protein - D3YVH2 CDS 3' 204 coding incomplete TSL:3

Ppp1r13l- ENSMUST00000132655.1 386 105aa ENSMUSP00000118309.1 Protein - D3YXE7 CDS 3' 203 coding incomplete TSL:3

Ppp1r13l- ENSMUST00000150448.7 340 No - lncRNA - - TSL:2 205 protein

Page 6 of 8 http://beta.alphaknockout.cyagen.net

38.78 kb Forward strand 19.35Mb 19.36Mb 19.37Mb 19.38Mb Genes (Comprehensive set... Ercc1-201 >protein coding Ppp1r13l-205 >processed transcript Ppp1r13l-203 >protein coding Ercc2-209 >processed transcript

Ercc1-202 >retained intron Ppp1r13l-204 >protein coding Ercc2-204 >processed transcript

Ercc1-204 >retained intron Ppp1r13l-202 >protein coding Ercc2-205 >processed transcript

Ercc1-203 >protein coding Ppp1r13l-201 >protein coding Ercc2-201 >protein coding

Ercc1-205 >nonsense mediated decay Ercc2-203 >protein coding

Ercc1-209 >protein coding Ercc2-202 >protein coding

Ercc1-207 >processed transcript Ercc2-206 >protein coding

Ercc1-206 >retained intron Mir343-201 >miRNA

Ercc1-210 >nonsense mediated decay

Ercc1-208 >retained intron

Contigs AC148988.5 > < AC118017.15 Genes < Cd3eap-201protein coding < Gm26852-201antisense (Comprehensive set...

< Cd3eap-202retained intron

Regulatory Build

19.35Mb 19.36Mb 19.37Mb 19.38Mb Reverse strand 38.78 kb

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Page 7 of 8 http://beta.alphaknockout.cyagen.net

Transcript: ENSMUST00000047621

17.32 kb Forward strand

Ppp1r13l-201 >protein coding

ENSMUSP00000047... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SH3-like domain superfamily

Ankyrin repeat-containing domain superfamily SMART Ankyrin repeat SH3 domain Prints SH3 domain Pfam Ankyrin repeat-containing domain

SH3 domain PROSITE profiles Ankyrin repeat SH3 domain

Ankyrin repeat-containing domain PANTHER PTHR24164:SF4

RelA-associated inhibitor Gene3D Ankyrin repeat-containing domain superfamily CDD RelA-associated inhibitor, SH3 domain

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

Variant Legend inframe insertion missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 824

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

Page 8 of 8