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Mouse Pop1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Pop1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Pop1 gene (NCBI Reference Sequence: NM_152894 ; Ensembl: ENSMUSG00000022325 ) is located on Mouse chromosome 15. 17 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 17 (Transcript: ENSMUST00000052290). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pop1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-365B13 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:

Exon 4 starts from about 9.92% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2480 bp, and the size of intron 5 for 3'-loxP site insertion: 1452 bp. The size of effective cKO region: ~2209 bp. The cKO 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 6 7 17 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pop1 Homology arm cKO region loxP site

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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(8709bp) | A(24.92% 2170) | C(22.01% 1917) | T(31.99% 2786) | G(21.08% 1836)

Note: The sequence of homologous arms and cKO region 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 3000 1 3000 3000 100.0% chr15 + 34501710 34504709 3000 browser details YourSeq 358 1327 2204 3000 89.0% chr15 - 102550445 102551051 607 browser details YourSeq 353 1327 1802 3000 90.1% chr10 - 62618568 62619002 435 browser details YourSeq 351 1302 1729 3000 93.8% chr1 - 125615241 125615669 429 browser details YourSeq 348 1327 1734 3000 93.6% chr8 + 122533816 122534215 400 browser details YourSeq 348 1327 1731 3000 93.6% chr10 + 69727993 69728389 397 browser details YourSeq 347 1313 1729 3000 92.2% chr14 - 24417488 24417892 405 browser details YourSeq 347 1327 1731 3000 93.5% chr7 + 45245807 45246201 395 browser details YourSeq 347 1327 1735 3000 92.9% chr6 + 38830595 38830997 403 browser details YourSeq 346 1327 1738 3000 94.0% chr13 - 83921027 83921447 421 browser details YourSeq 346 1195 1729 3000 90.1% chr14 + 16628636 16629096 461 browser details YourSeq 345 1327 2201 3000 93.1% chr4 + 143315475 143316350 876 browser details YourSeq 344 1327 1734 3000 92.4% chr9 - 59308771 59309169 399 browser details YourSeq 344 1327 1729 3000 94.0% chr9 - 23054219 23054615 397 browser details YourSeq 343 1327 1743 3000 92.3% chr9 - 75483705 75484105 401 browser details YourSeq 343 1327 1732 3000 92.6% chr2 + 3497468 3497866 399 browser details YourSeq 342 1327 1729 3000 92.7% chr5 - 17435201 17435592 392 browser details YourSeq 341 1327 1729 3000 92.8% chrX - 6858354 6858750 397 browser details YourSeq 341 1322 1727 3000 92.9% chr7 - 121178183 121178583 401 browser details YourSeq 341 1327 1725 3000 94.8% chr5 - 127729079 127729807 729

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% chr15 + 34506919 34509918 3000 browser details YourSeq 135 2122 2407 3000 91.0% chr4 - 128240423 128240704 282 browser details YourSeq 112 2125 2253 3000 96.0% chr6 + 73352688 73352818 131 browser details YourSeq 91 1033 2759 3000 94.3% chr9 + 73186646 73198326 11681 browser details YourSeq 86 2122 2230 3000 97.0% chr14 + 49215746 49215895 150 browser details YourSeq 86 2096 2225 3000 83.1% chr1 + 146696567 146696685 119 browser details YourSeq 79 2139 2223 3000 97.7% chr14 - 103261618 103261719 102 browser details YourSeq 75 2199 2340 3000 80.5% chr5 - 53185200 53185282 83 browser details YourSeq 71 2207 2399 3000 97.4% chr3 + 105626025 105626469 445 browser details YourSeq 70 2177 2302 3000 92.9% chr4 - 123071056 123071185 130 browser details YourSeq 69 977 1096 3000 77.4% chr4 + 62490114 62490229 116 browser details YourSeq 67 694 1098 3000 75.3% chr15 - 84051218 84051587 370 browser details YourSeq 67 2122 2366 3000 74.1% chr12 - 91577454 91577547 94 browser details YourSeq 67 977 1098 3000 78.7% chr6 + 37883401 37883507 107 browser details YourSeq 65 1010 1098 3000 93.3% chr11 + 98443755 98443845 91 browser details YourSeq 65 2163 2253 3000 96.0% chr1 + 14050814 14050977 164 browser details YourSeq 64 997 1098 3000 81.6% chr16 - 92192422 92192521 100 browser details YourSeq 63 980 1098 3000 74.8% chrX - 7938715 7938818 104 browser details YourSeq 63 2125 2235 3000 77.2% chr16 + 6449899 6449968 70 browser details YourSeq 63 1010 1098 3000 83.6% chr1 + 186804178 186804264 87

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Pop1 processing of precursor 1, ribonuclease P/MRP family, (S. cerevisiae) [ Mus musculus (house mouse) ] Gene ID: 67724, updated on 12-Aug-2019

Gene summary

Official Symbol Pop1 provided by MGI Official Full Name processing of precursor 1, ribonuclease P/MRP family, (S. cerevisiae) provided by MGI Primary source MGI:MGI:1914974 See related Ensembl:ENSMUSG00000022325 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 4932434G09Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 4.5), limb E14.5 (RPKM 3.2) and 27 other tissues See more Orthologs human all

Genomic context

Location: 15; 15 B3.1 See Pop1 in Genome Data Viewer

Exon count: 18

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (34495311..34530653)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (34425066..34460408)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 6 transcripts

Gene: Pop1 ENSMUSG00000022325

Description processing of precursor 1, ribonuclease P/MRP family, (S. cerevisiae) [Source:MGI Symbol;AccM: GI:1914974] Gene Synonyms 4932434G09Rik Location Chromosome 15: 34,495,304-34,530,648 forward strand. GRCm38:CM001008.2 About this gene This gene has 6 transcripts (splice variants), 205 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pop1-201 ENSMUST00000052290.13 3395 1045aa ENSMUSP00000052654.7 Protein coding CCDS27420 Q8K205 TSL:1 GENCODE basic APPRIS P4

Pop1-202 ENSMUST00000079028.5 3275 1015aa ENSMUSP00000078037.5 Protein coding CCDS27419 Q9D4G5 TSL:1 GENCODE basic APPRIS ALT2

Pop1-204 ENSMUST00000132427.1 794 No protein - Retained intron - - TSL:3

Pop1-205 ENSMUST00000153061.1 522 No protein - Retained intron - - TSL:2

Pop1-203 ENSMUST00000126223.1 392 No protein - Retained intron - - TSL:3

Pop1-206 ENSMUST00000155073.7 470 No protein - lncRNA - - TSL:3

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55.34 kb Forward strand 34.49Mb 34.50Mb 34.51Mb 34.52Mb 34.53Mb 34.54Mb Genes (Comprehensive set... Pop1-201 >protein coding Gm22208-201 >snRNA

Pop1-202 >protein coding

Pop1-203 >retained intron Pop1-205 >retained intron

Pop1-206 >lncRNA Gm49195-201 >processed pseudogene

Pop1-204 >retained intron

Contigs < AC126028.4 AC152940.2 > Genes < Rida-201protein coding (Comprehensive set...

< Rida-202retained intron

< Rida-204retained intron

< Rida-203lncRNA

Regulatory Build

34.49Mb 34.50Mb 34.51Mb 34.52Mb 34.53Mb 34.54Mb Reverse strand 55.34 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript pseudogene

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Transcript: ENSMUST00000052290

35.34 kb Forward strand

Pop1-201 >protein coding

ENSMUSP00000052... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Pop1, N-terminal POPLD domain PANTHER Ribonucleases P/MRP protein subunit Pop1

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1045

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

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