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Mouse Nlgn3 Knockout Project (CRISPR/Cas9)

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Mouse Nlgn3 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Nlgn3 Knockout Project (CRISPR/Cas9) Objective: To create a Nlgn3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Nlgn3 gene (NCBI Reference Sequence: NM_172932 ; Ensembl: ENSMUSG00000031302 ) is located on Mouse chromosome X. 7 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 7 (Transcript: ENSMUST00000065858). Exon 2~4 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: Homozygous null mice show impaired context and cued conditioning, hyperactivity, altered social behavior, less vocalization, smaller brains, and impaired olfaction. Males carrying a knock-in allele show impaired social interaction, and enhanced spatial learning and inhibitory synaptic transmission. Exon 2 starts from the coding region. Exon 2~4 covers 26.59% of the coding region. The size of effective KO region: ~7122 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 7 Legends Exon of mouse Nlgn3 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 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 2000 bp section downstream of Exon 4 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(18.6% 372) | C(26.4% 528) | T(28.95% 579) | G(26.05% 521) Note: The 2000 bp section upstream of Exon 2 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(2000bp) | A(25.9% 518) | C(27.8% 556) | T(29.65% 593) | G(16.65% 333) Note: The 2000 bp section downstream of Exon 4 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. Page 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chrX + 101299985 101301984 2000 browser details YourSeq 69 475 565 2000 92.9% chr14 - 19964611 19964958 348 browser details YourSeq 68 475 562 2000 94.9% chr1 + 35922733 35922869 137 browser details YourSeq 65 479 582 2000 78.0% chr8 + 99770124 99770204 81 browser details YourSeq 65 479 560 2000 92.2% chr14 + 64115905 64116029 125 browser details YourSeq 64 478 560 2000 93.4% chrX - 115071106 115071218 113 browser details YourSeq 64 479 562 2000 86.9% chr14 - 115207852 115207932 81 browser details YourSeq 64 475 562 2000 94.4% chr12 + 34609233 34609320 88 browser details YourSeq 62 520 592 2000 93.2% chr4 + 140494136 140494210 75 browser details YourSeq 60 479 562 2000 87.0% chr10 + 57465640 57465718 79 browser details YourSeq 59 479 560 2000 86.6% chrY - 80815975 80816057 83 browser details YourSeq 59 479 560 2000 86.6% chrY - 80015645 80015727 83 browser details YourSeq 59 479 560 2000 86.6% chrY - 65494325 65494407 83 browser details YourSeq 59 479 560 2000 86.6% chrY - 52896665 52896747 83 browser details YourSeq 57 479 560 2000 87.9% chrY + 36320169 36320257 89 browser details YourSeq 56 478 565 2000 80.6% chr12 - 27057131 27057209 79 browser details YourSeq 56 486 1007 2000 66.7% chr1 + 92700284 92700532 249 browser details YourSeq 55 480 1004 2000 65.1% chr17 - 10481083 10481154 72 browser details YourSeq 54 479 558 2000 93.3% chrY - 78316769 78316847 79 browser details YourSeq 53 490 573 2000 88.3% chr11 + 45582739 45582844 106 Note: The 2000 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 2000 1 2000 2000 100.0% chrX + 101308908 101310907 2000 browser details YourSeq 57 564 1173 2000 64.2% chr15 - 78071037 78071251 215 browser details YourSeq 55 1903 1984 2000 81.1% chr5 - 137355044 137355123 80 browser details YourSeq 54 862 1209 2000 69.4% chr1 - 89257573 89257747 175 browser details YourSeq 53 1790 1983 2000 82.7% chr12 - 76267965 76268157 193 browser details YourSeq 49 1918 1984 2000 86.6% chr18 - 7878714 7878780 67 browser details YourSeq 46 1912 1985 2000 81.1% chr12 - 100244024 100244097 74 browser details YourSeq 46 1919 1986 2000 83.9% chr1 - 138644324 138644391 68 browser details YourSeq 45 1921 1984 2000 90.0% chr8 - 72216852 72216914 63 browser details YourSeq 43 1453 1943 2000 80.0% chr11 - 119313519 119314001 483 browser details YourSeq 43 1919 1985 2000 82.1% chr12 + 76720201 76720267 67 browser details YourSeq 42 1912 1979 2000 80.9% chr13 - 44457283 44457350 68 browser details YourSeq 42 1921 1986 2000 84.0% chr1 + 136567081 136567145 65 browser details YourSeq 41 1921 1983 2000 82.6% chr10 - 42144554 42144616 63 browser details YourSeq 40 1912 1981 2000 78.6% chr1 - 104201862 104201931 70 browser details YourSeq 40 1921 1986 2000 80.4% chr4 + 41281268 41281333 66 browser details YourSeq 40 1921 1982 2000 82.3% chr1 + 21964761 21964822 62 browser details YourSeq 39 1921 1984 2000 81.0% chr7 + 101654668 101654733 66 browser details YourSeq 38 1921 1980 2000 81.7% chr12 + 24268236 24268295 60 browser details YourSeq 37 1921 1965 2000 91.2% chr15 - 31398540 31398584 45 Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Nlgn3 neuroligin 3 [ Mus musculus (house mouse) ] Gene ID: 245537, updated on 17-Sep-2019 Gene summary Official Symbol Nlgn3 provided by MGI Official Full Name neuroligin 3 provided by MGI Primary source MGI:MGI:2444609 See related Ensembl:ENSMUSG00000031302 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 NL3; HNL3; NLG3; A230085M13Rik Expression Biased expression in whole brain E14.5 (RPKM 20.3), CNS E18 (RPKM 19.2) and 11 other tissues See more Orthologs human all Genomic context Location: X; X D See Nlgn3 in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (101299179..101321350) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (98494520..98516689) Chromosome X - NC_000086.7 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Nlgn3 ENSMUSG00000031302 Description neuroligin 3 [Source:MGI Symbol;Acc:MGI:2444609] Gene Synonyms A230085M13Rik, HNL3, NL3, NLG3 Location Chromosome X: 101,299,168-101,325,963 forward strand. GRCm38:CM001013.2 About this gene This gene has 6 transcripts (splice variants), 233 orthologues, 25 paralogues, is a member of 1 Ensembl protein family and is associated with 35 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Nlgn3-201 ENSMUST00000065858.2 8465 825aa ENSMUSP00000066304.2 Protein coding CCDS30313 Q8BYM5 TSL:1 GENCODE basic APPRIS P2 Nlgn3-206 ENSMUST00000151528.7 8541 845aa ENSMUSP00000123283.1 Protein coding - A2AGI2 TSL:5 GENCODE basic APPRIS ALT1 Nlgn3-202 ENSMUST00000118111.7 8004 711aa ENSMUSP00000113863.1 Protein coding - A2AGI3 TSL:5 GENCODE basic Nlgn3-203 ENSMUST00000130555.7 1852 510aa ENSMUSP00000122213.1 Protein coding - A2AGI0 CDS 3' incomplete TSL:5 Nlgn3-205 ENSMUST00000147443.1 747 No protein - lncRNA - - TSL:5 Nlgn3-204 ENSMUST00000144860.1 636 No protein - lncRNA - - TSL:2 Page 7 of 9 https://www.alphaknockout.com 46.80 kb Forward strand 101.29Mb 101.30Mb 101.31Mb 101.32Mb 101.33Mb Genes (Comprehensive set... Med12-204 >protein coding Nlgn3-202 >protein coding Med12-203 >protein coding Nlgn3-203 >protein coding Med12-202 >protein coding Nlgn3-206 >protein coding Med12-201 >protein coding Nlgn3-201 >protein coding Med12-208 >retained intron Nlgn3-205 >lncRNA Nlgn3-204 >lncRNA Med12-205 >lncRNA Gm21986-201 >lncRNA Med12-206 >lncRNA Contigs AL683892.12 > Regulatory Build 101.29Mb 101.30Mb 101.31Mb 101.32Mb 101.33Mb Reverse strand 46.80 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 Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000065858 26.75 kb Forward strand Nlgn3-201 >protein coding ENSMUSP00000066... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily Alpha/Beta hydrolase fold Prints Neuroligin Pfam Carboxylesterase, type B PROSITE patterns Carboxylesterase type B, conserved site PANTHER Neuroligin-3 PTHR43903 Gene3D Alpha/Beta hydrolase fold All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend splice region variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 825 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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