https://www.alphaknockout.com

Mouse Pkd2l1 Knockout Project (CRISPR/Cas9)

Objective: To create a Pkd2l1 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Pkd2l1 gene (NCBI Reference Sequence: NM_181422 ; Ensembl: ENSMUSG00000037578 ) is located on Mouse chromosome 19. 15 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 15 (Transcript: ENSMUST00000042026). Exon 3~14 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 homozygous for a knock-out allele exhibit decreased chorda tympani nerve response to sour tastants.

Exon 3 starts from about 15.35% of the coding region. Exon 3~14 covers 82.98% of the coding region. The size of effective KO region: ~8612 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 3 4 5 6 7 8 9 10 11 12 13 14 15

Legends Exon of mouse Pkd2l1 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 3 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 604 bp section downstream of Exon 14 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(29.9% 598) | C(21.8% 436) | T(24.8% 496) | G(23.5% 470)

Note: The 2000 bp section upstream of Exon 3 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(604bp) | A(27.81% 168) | C(20.36% 123) | T(28.81% 174) | G(23.01% 139)

Note: The 604 bp section downstream of Exon 14 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% chr19 - 44157765 44159764 2000 browser details YourSeq 156 971 1433 2000 93.4% chr4 - 128501333 128906317 404985 browser details YourSeq 71 1364 1434 2000 100.0% chr14 - 46115664 46115734 71 browser details YourSeq 70 1364 1433 2000 100.0% chr5 - 101518608 101518677 70 browser details YourSeq 70 1364 1433 2000 100.0% chr19 - 53211615 53211684 70 browser details YourSeq 70 1364 1433 2000 100.0% chr15 - 54395292 54395361 70 browser details YourSeq 70 1364 1433 2000 100.0% chr1 - 31447503 31447572 70 browser details YourSeq 70 1364 1433 2000 100.0% chr19 + 10647746 10647815 70 browser details YourSeq 70 1364 1433 2000 100.0% chr11 + 10463802 10463871 70 browser details YourSeq 70 1364 1433 2000 100.0% chr11 + 10463808 10463877 70 browser details YourSeq 69 1362 1433 2000 98.7% chr14 - 61872176 61872251 76 browser details YourSeq 69 1364 1432 2000 100.0% chr1 - 188377430 188377498 69 browser details YourSeq 47 346 418 2000 82.2% chr17 + 36282250 36282322 73

Note: The 2000 bp section upstream of Exon 3 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 604 1 604 604 100.0% chr19 - 44148549 44149152 604 browser details YourSeq 23 107 132 604 96.0% chr13 + 107926621 107926655 35

Note: The 604 bp section downstream of Exon 14 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Pkd2l1 polycystic kidney disease 2-like 1 [ Mus musculus (house mouse) ] Gene ID: 329064, updated on 8-Oct-2019

Gene summary

Official Symbol Pkd2l1 provided by MGI Official Full Name polycystic kidney disease 2-like 1 provided by MGI Primary source MGI:MGI:1352448 See related Ensembl:ENSMUSG00000037578 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 PCL; Pkdl; PKD2L; TRPP3; BC046386; B830002B15 Expression Biased expression in testis adult (RPKM 2.9), cerebellum adult (RPKM 1.0) and 3 other tissues See more Orthologs human all

Genomic context

Location: 19 C3; 19 36.91 cM See Pkd2l1 in Genome Data Viewer Exon count: 26

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (44147637..44248328, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (44222127..44266932, complement)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 2 transcripts

Gene: Pkd2l1 ENSMUSG00000037578

Description polycystic kidney disease 2-like 1 [Source:MGI Symbol;Acc:MGI:1352448] Gene Synonyms PCL, PKD2L, Pkdl, TRPP3, polycystin-L Location Chromosome 19: 44,147,637-44,192,442 reverse strand. GRCm38:CM001012.2 About this gene This gene has 2 transcripts (splice variants), 195 orthologues, 10 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pkd2l1-201 ENSMUST00000042026.4 3321 760aa ENSMUSP00000045675.4 Protein coding CCDS29846 A2A259 TSL:1 GENCODE basic APPRIS P1

Pkd2l1-202 ENSMUST00000161357.1 3529 No protein - Retained intron - - TSL:2

64.81 kb Forward strand 44.14Mb 44.16Mb 44.18Mb 44.20Mb Contigs AC125101.3 >

Genes < Bloc1s2-204protein coding (Comprehensive set...

< Bloc1s2-201protein coding

< Bloc1s2-203lncRNA < Pkd2l1-201protein coding

< Bloc1s2-205lncRNA

< Bloc1s2-202lncRNA

< Pkd2l1-202retained intron

Regulatory Build

44.14Mb 44.16Mb 44.18Mb 44.20Mb Reverse strand 64.81 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000042026

< Pkd2l1-201protein coding

Reverse strand 44.81 kb

ENSMUSP00000045... Transmembrane heli... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF81324 EF-hand domain pair

Prints Polycystic kidney disease type 2 protein Pfam Polycystin cation channel, PKD1/PKD2 PF18109

PROSITE profiles EF-hand domain PANTHER PTHR10877:SF114

PTHR10877 Gene3D 1.10.287.70 1.10.238.10 1.20.5.340

Voltage-dependent channel domain superfamily

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

Variant Legend stop gained missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 760

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

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