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Mouse Homer3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Homer3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Homer3 gene (NCBI Reference Sequence: NM_001146153 ; Ensembl: ENSMUSG00000003573 ) is located on Mouse chromosome 8. 10 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000110124). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Homer3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-283L17 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: Homozygous mutants exhibit normal sensitivity to cocaine.

Exon 5 starts from about 28.23% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 3331 bp, and the size of intron 6 for 3'-loxP site insertion: 833 bp. The size of effective cKO region: ~1280 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 5 6 7 8 9 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Homer3 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(7780bp) | A(21.65% 1684) | C(24.97% 1943) | T(26.77% 2083) | G(26.61% 2070)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr8 + 70286145 70289144 3000 browser details YourSeq 242 581 2425 3000 92.7% chr4 - 141102430 141579351 476922 browser details YourSeq 181 2171 2449 3000 91.5% chr4 - 11312777 11313059 283 browser details YourSeq 173 1017 2399 3000 90.3% chr11 - 102048964 102071464 22501 browser details YourSeq 169 581 2407 3000 91.3% chr11 - 69886148 69967444 81297 browser details YourSeq 159 2037 2442 3000 95.0% chr3 - 95238821 95239371 551 browser details YourSeq 144 581 1092 3000 91.4% chr2 + 160658536 161136178 477643 browser details YourSeq 140 2097 2440 3000 84.0% chr1 - 91488419 91488613 195 browser details YourSeq 132 2294 2460 3000 90.8% chr6 + 86222968 86223136 169 browser details YourSeq 131 591 1128 3000 89.8% chr5 - 145077480 145078018 539 browser details YourSeq 131 2100 2442 3000 81.1% chr13 + 54635617 54635796 180 browser details YourSeq 128 581 1114 3000 82.0% chr18 + 56690596 56691111 516 browser details YourSeq 127 2294 2440 3000 93.2% chr4 - 11361649 11361795 147 browser details YourSeq 126 2296 2441 3000 93.2% chr6 - 18681693 18681838 146 browser details YourSeq 121 2294 2444 3000 89.3% chr1 + 144484881 144485030 150 browser details YourSeq 118 2103 2423 3000 80.6% chr5 + 37180902 37181040 139 browser details YourSeq 117 2098 2417 3000 80.8% chr2 + 147948174 147948308 135 browser details YourSeq 115 2150 2418 3000 89.7% chr16 - 33065131 33065504 374 browser details YourSeq 108 2307 2434 3000 93.0% chr10 - 38746410 38746543 134 browser details YourSeq 108 2294 2420 3000 93.0% chr10 + 4169421 4169549 129

Note: The 3000 bp section upstream of Exon 5 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% chr8 + 70290425 70293424 3000 browser details YourSeq 120 2155 2333 3000 89.8% chr17 - 4635934 4636111 178 browser details YourSeq 115 2151 2299 3000 89.2% chr10 + 39502749 39502898 150 browser details YourSeq 113 1967 2264 3000 89.7% chr18 + 14180983 14181514 532 browser details YourSeq 108 2146 2282 3000 90.5% chr15 - 12095185 12095342 158 browser details YourSeq 107 2146 2299 3000 87.3% chr17 + 70764749 70764898 150 browser details YourSeq 107 2171 2391 3000 88.7% chr11 + 48786791 48787326 536 browser details YourSeq 104 2147 2297 3000 89.4% chr14 - 39681397 39681552 156 browser details YourSeq 104 2167 2299 3000 90.0% chr13 - 110142647 110142781 135 browser details YourSeq 103 2146 2274 3000 90.7% chr11 - 115764223 115764362 140 browser details YourSeq 102 2175 2294 3000 94.8% chr15 + 99730770 99730903 134 browser details YourSeq 100 1967 2246 3000 91.2% chr12 - 24701594 24701957 364 browser details YourSeq 100 2141 2280 3000 86.4% chr11 - 88727126 88727276 151 browser details YourSeq 100 2171 2282 3000 95.6% chr7 + 29761332 29761455 124 browser details YourSeq 100 2147 2268 3000 91.9% chr16 + 91463050 91463183 134 browser details YourSeq 99 2147 2269 3000 91.1% chr12 - 77613102 77613236 135 browser details YourSeq 98 2146 2275 3000 88.5% chr13 - 106831461 106831602 142 browser details YourSeq 98 2161 2290 3000 89.6% chr11 - 104487556 104487696 141 browser details YourSeq 98 2143 2299 3000 88.9% chr1 + 178333185 178333662 478 browser details YourSeq 96 2171 2299 3000 91.4% chr8 - 72737859 72737991 133

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Homer3 homer scaffolding protein 3 [ Mus musculus (house mouse) ] Gene ID: 26558, updated on 12-Aug-2019

Gene summary

Official Symbol Homer3 provided by MGI Official Full Name homer scaffolding protein 3 provided by MGI Primary source MGI:MGI:1347359 See related Ensembl:ENSMUSG00000003573 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 AW146114 Expression Ubiquitous expression in cerebellum adult (RPKM 62.8), ovary adult (RPKM 38.6) and 27 other tissues See more Orthologs human all

Genomic context

Location: 8; 8 B3.3 See Homer3 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (70282880..70294361)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (72806898..72818260)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 8 transcripts

Gene: Homer3 ENSMUSG00000003573

Description homer scaffolding protein 3 [Source:MGI Symbol;Acc:MGI:1347359] Location Chromosome 8: 70,282,827-70,294,361 forward strand. GRCm38:CM001001.2 About this gene This gene has 8 transcripts (splice variants), 254 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Homer3-206 ENSMUST00000140212.7 2608 356aa ENSMUSP00000117033.1 Protein coding CCDS22363 Q99JP6 TSL:1 GENCODE basic APPRIS P3

Homer3-203 ENSMUST00000110124.8 2445 359aa ENSMUSP00000105751.2 Protein coding CCDS52569 Q99JP6 TSL:5 GENCODE basic APPRIS ALT1

Homer3-201 ENSMUST00000003669.7 2323 359aa ENSMUSP00000003669.7 Protein coding CCDS52569 Q99JP6 TSL:1 GENCODE basic APPRIS ALT1

Homer3-202 ENSMUST00000087467.11 880 122aa ENSMUSP00000084735.5 Protein coding - Q501M9 TSL:1 GENCODE basic

Homer3-204 ENSMUST00000135368.1 583 93aa ENSMUSP00000137560.1 Protein coding - J3QQ00 CDS 5' incomplete TSL:3

Homer3-207 ENSMUST00000143528.7 852 No protein - Retained intron - - TSL:3

Homer3-205 ENSMUST00000135692.1 805 No protein - Retained intron - - TSL:3

Homer3-208 ENSMUST00000155711.1 678 No protein - Retained intron - - TSL:2

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31.54 kb Forward strand

70.28Mb 70.29Mb 70.30Mb Genes (Comprehensive set... Sugp2-211 >lncRNA Homer3-206 >protein coding Cope-208 >nonsense mediated decay

Sugp2-203 >lncRNA Homer3-202 >protein coding Homer3-205 >retained intron Cope-205 >retained intron

Homer3-203 >protein coding Cope-204 >retained intron

Homer3-208 >retained intron Homer3-204 >protein coding Cope-203 >retained intron

Homer3-201 >protein coding Cope-201 >protein coding

Homer3-207 >retained intron Cope-206 >protein coding

Contigs AC124327.9 > < AC158553.13 Genes < Ddx49-201protein coding (Comprehensive set...

< Ddx49-205retained intron

< Ddx49-203retained intron < Ddx49-202retained intron

< Ddx49-204retained intron

Regulatory Build

70.28Mb 70.29Mb 70.30Mb Reverse strand 31.54 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000110124

11.36 kb Forward strand

Homer3-203 >protein coding

ENSMUSP00000105... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF50729 SMART WH1/EVH1 domain Pfam WH1/EVH1 domain PROSITE profiles WH1/EVH1 domain PANTHER PTHR10918

PTHR10918:SF4 Gene3D PH-like domain superfamily CDD cd01206

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 359

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

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