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Mouse Syngr1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Syngr1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Syngr1 gene (NCBI Reference Sequence: NM_207708 ; Ensembl: ENSMUSG00000022415 ) is located on Mouse chromosome 15. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000009727). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Syngr1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-62P17 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 knock-out allele exhibit a moderate decrease in post-tetanic potentiation.

Exon 2 starts from about 14.25% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 18951 bp, and the size of intron 2 for 3'-loxP site insertion: 917 bp. The size of effective cKO region: ~738 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 2 3 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Syngr1 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. 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 GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7238bp) | A(22.78% 1649) | C(27.88% 2018) | T(26.1% 1889) | G(23.24% 1682)

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 + 80107195 80110194 3000 browser details YourSeq 1354 1235 3000 3000 90.9% chr5 - 24898210 24899989 1780 browser details YourSeq 1351 1246 3000 3000 89.7% chr1 + 177883996 177885775 1780 browser details YourSeq 1350 1257 3000 3000 90.5% chr11 + 95320391 95322177 1787 browser details YourSeq 1332 1242 3000 3000 90.6% chr3 - 151206472 151208244 1773 browser details YourSeq 1331 1242 2992 3000 89.8% chr13 - 76601599 76603374 1776 browser details YourSeq 1327 1271 3000 3000 90.3% chr19 + 48374685 48376437 1753 browser details YourSeq 1326 1242 3000 3000 90.5% chr18 - 45045720 45047501 1782 browser details YourSeq 1324 1242 3000 3000 90.0% chr11 - 5651315 5653088 1774 browser details YourSeq 1314 1241 3000 3000 90.4% chr13 + 92282243 92284025 1783 browser details YourSeq 1309 1305 3000 3000 90.1% chr3 - 52846073 52847806 1734 browser details YourSeq 1309 1244 3000 3000 89.3% chr6 + 36503717 36505485 1769 browser details YourSeq 1309 1244 3000 3000 90.5% chr14 + 16369831 16371608 1778 browser details YourSeq 1307 1269 3000 3000 89.8% chr14 + 95941580 96355300 413721 browser details YourSeq 1306 1305 3000 3000 90.3% chr10 - 119402699 119404431 1733 browser details YourSeq 1286 1347 3000 3000 90.8% chr16 + 63024475 63026179 1705 browser details YourSeq 1284 1319 3000 3000 90.3% chr18 + 12665396 12667115 1720 browser details YourSeq 1280 1235 3000 3000 90.5% chr16 - 93673276 93675499 2224 browser details YourSeq 1280 1299 3000 3000 90.2% chr10 + 66559526 66779653 220128 browser details YourSeq 1280 1315 3000 3000 90.4% chr1 + 88522051 88523771 1721

Note: The 3000 bp section upstream of Exon 2 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 + 80110933 80113932 3000 browser details YourSeq 171 1289 1872 3000 91.4% chr13 + 37781399 38031346 249948 browser details YourSeq 139 1334 1860 3000 93.1% chr14 - 29982909 29983540 632 browser details YourSeq 131 1690 1857 3000 93.4% chr19 - 44970437 44970850 414 browser details YourSeq 131 1702 1870 3000 91.7% chr17 + 83579252 83579437 186 browser details YourSeq 129 1251 1825 3000 90.6% chr9 + 27138911 27139498 588 browser details YourSeq 129 1694 1870 3000 93.3% chr16 + 93843289 93843472 184 browser details YourSeq 128 1703 1877 3000 91.1% chr17 + 29372458 29372661 204 browser details YourSeq 127 1691 1861 3000 92.6% chr12 - 99431247 99431424 178 browser details YourSeq 126 1691 1863 3000 92.6% chr1 - 182077915 182078097 183 browser details YourSeq 125 1705 1863 3000 91.9% chr3 - 95462014 95462179 166 browser details YourSeq 125 1691 1857 3000 90.4% chr2 - 51454897 51455077 181 browser details YourSeq 125 1691 1860 3000 92.6% chr19 - 4385919 4386421 503 browser details YourSeq 123 1706 1860 3000 92.4% chr2 - 80525038 80525206 169 browser details YourSeq 123 1694 1860 3000 91.8% chr17 - 53545813 53545989 177 browser details YourSeq 123 2644 2784 3000 92.2% chr3 + 129866830 129866969 140 browser details YourSeq 123 1694 1860 3000 91.3% chr10 + 67347471 67347649 179 browser details YourSeq 122 2646 2784 3000 94.9% chr14 - 99226574 99226721 148 browser details YourSeq 122 1694 1854 3000 91.8% chr1 - 187980006 187980176 171 browser details YourSeq 122 2641 2784 3000 93.1% chr2 + 122122686 122122832 147

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Syngr1 synaptogyrin 1 [ Mus musculus (house mouse) ] Gene ID: 20972, updated on 17-Sep-2019

Gene summary

Official Symbol Syngr1 provided by MGI Official Full Name synaptogyrin 1 provided by MGI Primary source MGI:MGI:1328323 See related Ensembl:ENSMUSG00000022415 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 p29; R75468; Syngr1b Expression Biased expression in cerebellum adult (RPKM 56.1), cortex adult (RPKM 51.4) and 14 other tissues See more Orthologs human all

Genomic context

Location: 15; 15 E1 See Syngr1 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (80091334..80119501)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (79921764..79949931)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 4 transcripts

Gene: Syngr1 ENSMUSG00000022415

Description synaptogyrin 1 [Source:MGI Symbol;Acc:MGI:1328323] Gene Synonyms Syngr1b, p29 Location Chromosome 15: 80,091,334-80,119,501 forward strand. GRCm38:CM001008.2 About this gene This gene has 4 transcripts (splice variants), 225 orthologues, 3 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

Syngr1-201 ENSMUST00000009727.11 4180 234aa ENSMUSP00000009727.5 Protein coding CCDS27657 O55100 TSL:1 GENCODE basic APPRIS P4

Syngr1-202 ENSMUST00000009728.12 819 191aa ENSMUSP00000009728.6 Protein coding CCDS27658 O55100 Q3U6D7 TSL:1 GENCODE basic APPRIS ALT1

Syngr1-203 ENSMUST00000135727.1 576 128aa ENSMUSP00000121716.1 Protein coding - F6PZA3 CDS 5' incomplete TSL:3

Syngr1-204 ENSMUST00000143928.1 479 111aa ENSMUSP00000120696.1 Protein coding - D3YWY9 CDS 3' incomplete TSL:3

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48.17 kb Forward strand

80.09Mb 80.10Mb 80.11Mb 80.12Mb Genes (Comprehensive set... Syngr1-202 >protein coding Syngr1-203 >protein coding

Syngr1-201 >protein coding

Syngr1-204 >protein coding

Contigs AC161199.4 > Genes < Rpl3-201protein coding < Gm24204-201snRNA (Comprehensive set...

< Rpl3-206retained intron

< Rpl3-204retained intron

< Rpl3-205nonsense mediated decay

< Rpl3-202protein coding

< Rpl3-203retained intron

< Snord43-201miRNA

Regulatory Build

80.09Mb 80.10Mb 80.11Mb 80.12Mb Reverse strand 48.17 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000009727

28.16 kb Forward strand

Syngr1-201 >protein coding

ENSMUSP00000009... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam Marvel domain

PROSITE profiles Marvel domain

PIRSF Synaptogyrin PANTHER PTHR10838:SF7

Synaptogyrin

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

Variant Legend

synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 200 234

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

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