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Mouse Pacsin2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Pacsin2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Pacsin2 gene (NCBI Reference Sequence: NM_001159509 ; Ensembl: ENSMUSG00000016664 ) is located on Mouse chromosome 15. 11 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000171436). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pacsin2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-63O19 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: Mice homozygous for a null allele exhibit reduced running endurance, distance, and speed with impaired fetal cardiomyocyte electrophysiology.

Exon 3 starts from about 4.18% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 9209 bp, and the size of intron 3 for 3'-loxP site insertion: 1842 bp. The size of effective cKO region: ~657 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 4 11 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pacsin2 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(7157bp) | A(24.17% 1730) | C(23.54% 1685) | T(26.85% 1922) | G(25.43% 1820)

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 - 83392908 83395907 3000 browser details YourSeq 161 84 360 3000 85.9% chr1 + 90771727 90772021 295 browser details YourSeq 158 86 360 3000 83.0% chr12 - 17087165 17087452 288 browser details YourSeq 155 85 360 3000 81.2% chr13 - 103323273 103323549 277 browser details YourSeq 155 87 360 3000 89.8% chr1 + 37888654 37888940 287 browser details YourSeq 152 85 360 3000 82.8% chr11 + 113535496 113535776 281 browser details YourSeq 151 84 360 3000 83.3% chr1 - 179138658 179138935 278 browser details YourSeq 148 84 361 3000 79.9% chr1 + 132672229 132672508 280 browser details YourSeq 143 146 361 3000 87.5% chr9 - 63901288 63901520 233 browser details YourSeq 139 84 359 3000 80.6% chr1 + 13135358 13135629 272 browser details YourSeq 133 82 361 3000 82.8% chr8 + 15602819 15603086 268 browser details YourSeq 132 84 368 3000 91.8% chr11 - 82678275 82854057 175783 browser details YourSeq 131 116 349 3000 82.9% chr15 - 86121095 86121344 250 browser details YourSeq 131 86 361 3000 84.7% chr11 + 77422769 77423046 278 browser details YourSeq 130 87 357 3000 86.7% chr12 - 57671815 57672098 284 browser details YourSeq 129 84 362 3000 85.0% chr15 - 7093401 7093681 281 browser details YourSeq 128 84 351 3000 84.9% chr10 + 43692718 43692984 267 browser details YourSeq 127 86 361 3000 75.3% chr7 + 34822719 34822975 257 browser details YourSeq 126 84 361 3000 82.5% chr2 + 128760909 128761183 275 browser details YourSeq 126 86 351 3000 88.0% chr10 + 123851309 123851578 270

Note: The 3000 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 3000 1 3000 3000 100.0% chr15 - 83389251 83392250 3000 browser details YourSeq 144 2649 2863 3000 84.7% chr8 + 22447772 22447987 216 browser details YourSeq 139 2532 2861 3000 88.2% chr19 + 56933601 56934196 596 browser details YourSeq 139 2643 2847 3000 84.3% chr11 + 11585075 11585278 204 browser details YourSeq 137 2649 2861 3000 82.0% chr7 + 142958750 142958960 211 browser details YourSeq 137 2649 2860 3000 86.7% chr14 + 66250956 66251168 213 browser details YourSeq 134 2649 2861 3000 86.5% chr11 - 11732411 11732627 217 browser details YourSeq 133 2649 2867 3000 90.5% chr4 + 119216162 119216384 223 browser details YourSeq 132 2647 2860 3000 86.0% chr18 + 20968148 20968365 218 browser details YourSeq 131 2669 2852 3000 88.0% chr10 - 55518123 55518313 191 browser details YourSeq 130 2661 2857 3000 89.3% chr7 + 144804109 144804309 201 browser details YourSeq 129 2646 2863 3000 85.0% chr2 - 116942825 116943041 217 browser details YourSeq 127 2653 2861 3000 84.0% chrX + 13499685 13499855 171 browser details YourSeq 127 2649 2863 3000 84.4% chr1 + 138463510 138463726 217 browser details YourSeq 125 2643 2837 3000 89.5% chr15 - 101007812 101008012 201 browser details YourSeq 125 2671 2863 3000 86.1% chr12 - 70038901 70039091 191 browser details YourSeq 124 2650 2847 3000 82.9% chr10 - 117468248 117468440 193 browser details YourSeq 123 2670 2861 3000 86.0% chr12 - 42204927 42205116 190 browser details YourSeq 123 2649 2838 3000 86.5% chr4 + 123231823 123232013 191 browser details YourSeq 122 2662 2852 3000 83.8% chr14 + 58017196 58017393 198

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Pacsin2 protein kinase C and casein kinase substrate in neurons 2 [ Mus musculus (house mouse) ] Gene ID: 23970, updated on 1-Oct-2019

Gene summary

Official Symbol Pacsin2 provided by MGI Official Full Name protein kinase C and casein kinase substrate in neurons 2 provided by MGI Primary source MGI:MGI:1345153 See related Ensembl:ENSMUSG00000016664 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 AI197433 Expression Ubiquitous expression in heart adult (RPKM 34.6), kidney adult (RPKM 33.9) and 28 other tissues See more Orthologs human all

Genomic context

Location: 15; 15 E1 See Pacsin2 in Genome Data Viewer

Exon count: 17

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (83375607..83464610, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (83206037..83295036, complement)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 11 transcripts

Gene: Pacsin2 ENSMUSG00000016664

Description protein kinase C and casein kinase substrate in neurons 2 [Source:MGI Symbol;Acc:MGI:1345153] Gene Synonyms Syndapin II Location Chromosome 15: 83,375,607-83,464,606 reverse strand. GRCm38:CM001008.2 About this gene This gene has 11 transcripts (splice variants), 198 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pacsin2- ENSMUST00000171436.7 3693 486aa ENSMUSP00000131504.1 Protein coding CCDS27701 Q3TDA7 TSL:5 203 Q9WVE8 GENCODE basic APPRIS P1

Pacsin2- ENSMUST00000165095.8 3186 486aa ENSMUSP00000130098.1 Protein coding CCDS27701 Q3TDA7 TSL:1 202 Q9WVE8 GENCODE basic APPRIS P1

Pacsin2- ENSMUST00000231184.1 3103 486aa ENSMUSP00000155334.1 Protein coding CCDS27701 Q3TDA7 GENCODE basic 210 Q9WVE8 APPRIS P1

Pacsin2- ENSMUST00000230679.1 3083 486aa ENSMUSP00000155481.1 Protein coding CCDS27701 Q3TDA7 GENCODE basic 206 Q9WVE8 APPRIS P1

Pacsin2- ENSMUST00000056177.6 2795 486aa ENSMUSP00000058320.6 Protein coding CCDS27701 Q3TDA7 TSL:1 201 Q9WVE8 GENCODE basic APPRIS P1

Pacsin2- ENSMUST00000230030.1 857 272aa ENSMUSP00000155333.1 Protein coding - A0A2R8W6S4 CDS 5' 205 incomplete

Pacsin2- ENSMUST00000231946.1 823 180aa ENSMUSP00000155925.1 Protein coding - A0A338P6P7 CDS 5' 211 incomplete

Pacsin2- ENSMUST00000230816.1 765 191aa ENSMUSP00000155068.1 Protein coding - Q3UP40 CDS 3' 207 incomplete

Pacsin2- ENSMUST00000229337.1 640 124aa ENSMUSP00000155845.1 Protein coding - A0A2R8W750 CDS 3' 204 incomplete

Pacsin2- ENSMUST00000230960.1 639 No - Retained - - - 208 protein intron

Pacsin2- ENSMUST00000231043.1 635 No - lncRNA - - - 209 protein

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109.00 kb Forward strand

83.38Mb 83.40Mb 83.42Mb 83.44Mb 83.46Mb Contigs AC165945.7 > < AL583889.8

Genes (Comprehensive set... < 1700001L05Rik-203lncRNA < Pacsin2-207protein coding

< 1700001L05Rik-204lncRNA < Pacsin2-204protein coding

< 1700001L05Rik-201lncRNA < Gm5417-201processed pseudogene

< Pacsin2-203protein coding

< Pacsin2-202protein coding

< Pacsin2-201protein coding

< Pacsin2-206protein coding

< Pacsin2-210protein coding

< Pacsin2-211protein coding

< Pacsin2-205protein coding

< Pacsin2-209lncRNA

< Pacsin2-208retained intron

Regulatory Build

83.38Mb 83.40Mb 83.42Mb 83.44Mb 83.46Mb Reverse strand 109.00 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene pseudogene

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Transcript: ENSMUST00000171436

< Pacsin2-203protein coding

Reverse strand 89.00 kb

ENSMUSP00000131... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily AH/BAR domain superfamily SH3-like domain superfamily

SMART FCH domain SH3 domain

Prints SH3 domain Pfam FCH domain SH3 domain

PROSITE profiles F-BAR domain SH3 domain

PANTHER Protein kinase C and casein kinase substrate in neurons protein 2

PTHR23065 Gene3D AH/BAR domain superfamily 2.30.30.40

CDD PACSIN2, F-BAR domain PACSIN1/PACSIN2, SH3 domain

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 486

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

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