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Mouse Gcsh Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Gcsh gene (NCBI Reference Sequence: NM_026572 ; Ensembl: ENSMUSG00000034424 ) is located on Mouse chromosome 8. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000040484). 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 Gcsh gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-254F5 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 2 starts from about 27.45% 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: 4084 bp, and the size of intron 2 for 3'-loxP site insertion: 1747 bp. The size of effective cKO region: ~580 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 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Gcsh 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(7080bp) | A(24.68% 1747) | C(22.23% 1574) | T(29.65% 2099) | G(23.45% 1660)

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% chr8 - 116989454 116992453 3000 browser details YourSeq 344 610 2103 3000 93.5% chr11 - 62474708 62485100 10393 browser details YourSeq 300 1532 2075 3000 95.0% chr10 - 41862448 42602771 740324 browser details YourSeq 278 1544 2093 3000 93.0% chr12 - 24688318 24777663 89346 browser details YourSeq 268 1567 2076 3000 88.7% chr11 - 86678070 86678413 344 browser details YourSeq 261 1573 2072 3000 94.9% chr1 + 72818257 73385633 567377 browser details YourSeq 258 1582 2076 3000 90.5% chr7 - 141417135 141417594 460 browser details YourSeq 246 1594 2102 3000 90.1% chr10 - 128554390 128554860 471 browser details YourSeq 243 1599 2099 3000 94.9% chr10 + 80213921 80591977 378057 browser details YourSeq 231 1564 2072 3000 86.4% chr14 - 50889829 50890142 314 browser details YourSeq 231 1558 2037 3000 89.7% chr10 - 80291359 80291783 425 browser details YourSeq 216 1629 2076 3000 95.4% chr10 + 59945373 59945910 538 browser details YourSeq 184 1577 2059 3000 91.8% chrX + 159438595 159439154 560 browser details YourSeq 178 1683 2091 3000 95.4% chr8 + 70666101 70725697 59597 browser details YourSeq 165 1662 2072 3000 96.2% chr11 + 86986305 86986727 423 browser details YourSeq 159 1913 2089 3000 97.1% chr10 + 95946841 95947027 187 browser details YourSeq 156 1897 2080 3000 94.4% chr11 - 73162005 73162213 209 browser details YourSeq 156 1915 2076 3000 98.8% chr16 + 32410209 32410372 164 browser details YourSeq 153 1917 2075 3000 98.2% chr16 - 18726413 18726571 159 browser details YourSeq 153 1915 2083 3000 93.3% chrX + 83062211 83062375 165

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% chr8 - 116985874 116988873 3000 browser details YourSeq 116 2674 2837 3000 91.5% chr18 + 78051771 78051934 164 browser details YourSeq 103 2711 2837 3000 92.7% chr15 - 83163588 83163731 144 browser details YourSeq 98 2714 2837 3000 92.4% chr5 - 151005020 151005153 134 browser details YourSeq 97 2726 2859 3000 93.7% chr1 + 82780544 82780696 153 browser details YourSeq 96 2677 2837 3000 80.9% chr9 + 13777582 13777704 123 browser details YourSeq 95 2719 2837 3000 91.4% chr17 - 41371738 41371858 121 browser details YourSeq 95 2726 2837 3000 92.9% chr16 - 45266587 45266700 114 browser details YourSeq 95 2718 2859 3000 90.7% chr14 - 92760888 92761032 145 browser details YourSeq 95 2717 2837 3000 91.5% chr7 + 16955701 16956204 504 browser details YourSeq 94 2727 2837 3000 93.6% chr8 - 123100235 123100347 113 browser details YourSeq 92 2721 2837 3000 89.8% chr3 + 119934560 119934678 119 browser details YourSeq 91 2721 2837 3000 91.2% chr17 + 74320322 74320443 122 browser details YourSeq 91 2710 2837 3000 90.4% chr1 + 93846351 93846547 197 browser details YourSeq 89 2732 2837 3000 93.4% chr5 - 95807233 95807345 113 browser details YourSeq 89 2720 2859 3000 87.0% chr15 - 102876210 102876392 183 browser details YourSeq 88 2726 2837 3000 90.2% chr10 - 111368547 111368664 118 browser details YourSeq 88 2729 2855 3000 86.7% chr2 + 127902448 127902593 146 browser details YourSeq 86 2721 2823 3000 93.3% chr18 - 38127899 38128225 327 browser details YourSeq 85 2721 2823 3000 91.3% chr15 + 100515871 100515973 103

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: Gcsh glycine cleavage system protein H (aminomethyl carrier) [ Mus musculus (house mouse) ] Gene ID: 68133, updated on 12-Aug-2019

Gene summary

Official Symbol Gcsh provided by MGI Official Full Name glycine cleavage system protein H (aminomethyl carrier) provided by MGI Primary source MGI:MGI:1915383 See related Ensembl:ENSMUSG00000034424 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 1100001L02Rik; 5730591C18Rik Expression Ubiquitous expression in placenta adult (RPKM 73.9), kidney adult (RPKM 41.7) and 27 other tissues See more Orthologs human all

Genomic context

Location: 8; 8 E1 See Gcsh in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (116981967..116993498, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (119505728..119517349, complement)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 2 transcripts

Gene: Gcsh ENSMUSG00000034424

Description glycine cleavage system protein H (aminomethyl carrier) [Source:MGI Symbol;Acc:MGI:1915383] Gene Synonyms 1100001L02Rik, 5730591C18Rik Location Chromosome 8: 116,981,810-116,993,537 reverse strand. GRCm38:CM001001.2 About this gene This gene has 2 transcripts (splice variants), 217 orthologues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Gcsh-201 ENSMUST00000040484.5 1443 170aa ENSMUSP00000037131.5 Protein coding CCDS22696 Q91WK5 TSL:1 GENCODE basic APPRIS P1

Gcsh-202 ENSMUST00000162548.1 384 No protein - lncRNA - - TSL:3

31.73 kb Forward strand 116.98Mb 116.99Mb 117.00Mb Contigs < AC162858.6

Genes (Comprehensive set... < 1700030J22Rik-201protein coding < Gcsh-201protein coding < Pkd1l2-201protein coding

< 1700030J22Rik-202protein coding < Gcsh-202lncRNA < Pkd1l2-202protein coding

Regulatory Build

116.98Mb 116.99Mb 117.00Mb Reverse strand 31.73 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000040484

< Gcsh-201protein coding

Reverse strand 11.73 kb

ENSMUSP00000037... PDB-ENSP mappings TIGRFAM Glycine cleavage system H-protein, subgroup Superfamily Single hybrid motif Pfam Glycine cleavage system H-protein/Simiate PROSITE profiles Biotin/lipoyl attachment PROSITE patterns 2-oxo acid dehydrogenase, lipoyl-binding site PANTHER PTHR11715:SF28

Glycine cleavage system H-protein HAMAP Glycine cleavage system H-protein Gene3D 2.40.50.100 CDD Glycine cleavage system H-protein/Simiate

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 170

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

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