https://www.alphaknockout.com

Mouse Suclg2 Knockout Project (CRISPR/Cas9)

Objective: To create a Suclg2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Suclg2 (NCBI Reference Sequence: NM_011507 ; Ensembl: ENSMUSG00000061838 ) is located on Mouse 6. 11 are identified, with the ATG start codon in 1 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000204224). Exon 2 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 knockout with a gene trap is embryonic lethal. Heterozygosity has a mild effect on mitochondrial respiration.

Exon 2 starts from about 6.77% of the coding region. Exon 2 covers 10.93% of the coding region. The size of effective KO region: ~142 bp. The KO 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 2 11

Legends Exon of mouse Suclg2 Knockout region

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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 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(23.0% 460) | C(21.05% 421) | T(35.45% 709) | G(20.5% 410)

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(26.15% 523) | C(19.15% 383) | T(31.35% 627) | G(23.35% 467)

Note: The 2000 bp section downstream 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr6 - 95655600 95657599 2000 browser details YourSeq 259 419 1560 2000 83.0% chr7 - 135320679 135321790 1112 browser details YourSeq 166 835 1506 2000 89.2% chr12 - 51329023 51329790 768 browser details YourSeq 166 435 848 2000 87.5% chr11 + 19896201 19896694 494 browser details YourSeq 163 386 1144 2000 90.2% chr11 + 43153840 43154629 790 browser details YourSeq 161 565 1155 2000 86.8% chrX + 51950431 51951025 595 browser details YourSeq 157 364 759 2000 88.3% chrX + 101856134 101856557 424 browser details YourSeq 157 496 965 2000 82.5% chr14 + 63012575 63013027 453 browser details YourSeq 149 592 1324 2000 86.3% chr14 + 17799229 17799976 748 browser details YourSeq 137 432 785 2000 88.8% chrX + 138790396 138790754 359 browser details YourSeq 132 655 1318 2000 88.6% chr15 - 55154221 55154933 713 browser details YourSeq 129 386 759 2000 86.0% chr12 + 73143453 73143834 382 browser details YourSeq 123 719 1186 2000 78.9% chrX - 168025577 168026068 492 browser details YourSeq 123 1042 1468 2000 82.5% chr14 + 21910523 21910952 430 browser details YourSeq 120 500 687 2000 88.0% chr13 - 62947972 62948178 207 browser details YourSeq 120 544 936 2000 78.1% chrX + 146316310 146316684 375 browser details YourSeq 118 609 1118 2000 84.0% chr2 - 79055348 79055841 494 browser details YourSeq 114 379 646 2000 88.0% chr13 + 73045940 73046214 275 browser details YourSeq 112 561 1083 2000 87.4% chrX - 104939968 104940496 529 browser details YourSeq 112 559 1098 2000 87.4% chr12 - 96745171 96745741 571

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% chr6 - 95653458 95655457 2000 browser details YourSeq 61 991 1083 2000 87.7% chr6 - 82439877 82851656 411780 browser details YourSeq 61 962 1119 2000 87.5% chr14 - 18277152 18277320 169 browser details YourSeq 47 1016 1081 2000 87.4% chr5 - 106198613 106198681 69 browser details YourSeq 45 987 1083 2000 79.6% chr16 - 92496764 92496847 84 browser details YourSeq 45 997 1083 2000 74.7% chr18 + 21070316 21070399 84 browser details YourSeq 38 991 1048 2000 95.2% chr5 - 108440707 108440765 59 browser details YourSeq 37 989 1047 2000 97.5% chr16 + 55817174 55817235 62 browser details YourSeq 35 1030 1082 2000 83.1% chr8 + 70097006 70097058 53 browser details YourSeq 35 994 1047 2000 94.9% chr19 + 59188035 59188089 55 browser details YourSeq 33 1031 1095 2000 77.8% chr5 + 137812206 137812263 58 browser details YourSeq 33 1028 1165 2000 57.2% chr12 + 104487795 104487896 102 browser details YourSeq 33 1030 1080 2000 82.4% chr1 + 89445101 89445151 51 browser details YourSeq 32 1033 1090 2000 77.6% chr3 - 35844941 35844998 58 browser details YourSeq 32 1029 1082 2000 79.7% chr2 - 155987869 155987922 54 browser details YourSeq 32 1025 1083 2000 97.2% chr17 + 27387039 27387118 80 browser details YourSeq 32 1028 1114 2000 94.5% chr15 + 30296740 30296827 88 browser details YourSeq 30 1031 1080 2000 80.0% chr9 + 56964602 56964651 50 browser details YourSeq 30 1029 1098 2000 74.2% chr7 + 19670650 19670720 71 browser details YourSeq 29 1030 1100 2000 70.5% chr17 - 86557191 86557261 71

Note: The 2000 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: Suclg2 succinate-Coenzyme A ligase, GDP-forming, beta subunit [ Mus musculus (house mouse) ] Gene ID: 20917, updated on 12-Aug-2019

Gene summary

Official Symbol Suclg2 provided by MGI Official Full Name succinate-Coenzyme A ligase, GDP-forming, beta subunit provided by MGI Primary source MGI:MGI:1306824 See related Ensembl:ENSMUSG00000061838 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 G-SCS; GTPSCS; AF171077; AW556404; D6Wsu120e; SCS-betaG Expression Ubiquitous expression in adult (RPKM 29.5), liver adult (RPKM 17.1) and 27 other tissuesS ee more Orthologs human all

Genomic context

Location: 6 D2; 6 44.63 cM See Suclg2 in Genome Data Viewer Exon count: 13

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (95473009..95718858, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (95424128..95668831, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 5 transcripts

Gene: Suclg2 ENSMUSG00000061838

Description succinate-Coenzyme A ligase, GDP-forming, beta subunit [Source:MGI Symbol;Acc:MGI:1306824] Gene Synonyms D6Wsu120e Location Chromosome 6: 95,473,009-95,718,800 reverse strand. GRCm38:CM000999.2 About this gene This gene has 5 transcripts (splice variants), 213 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Suclg2-204 ENSMUST00000204224.2 3367 433aa ENSMUSP00000144827.1 Protein coding CCDS20381 Q9Z2I8 TSL:1 GENCODE basic APPRIS P1

Suclg2-201 ENSMUST00000079847.7 2518 384aa ENSMUSP00000078774.6 Protein coding CCDS85100 Q9Z2I8 TSL:1 GENCODE basic

Suclg2-205 ENSMUST00000204567.1 385 83aa ENSMUSP00000145471.1 Protein coding - A0A0N4SWD1 CDS 3' incomplete TSL:3

Suclg2-203 ENSMUST00000203109.1 961 No protein - Retained intron - - TSL:2

Suclg2-202 ENSMUST00000203071.1 622 No protein - lncRNA - - TSL:5

265.79 kb Forward strand 95.5Mb 95.6Mb 95.7Mb Contigs < AC125225.4 < AC135018.3 AC121612.2 > (Comprehensive set... < Suclg2-204protein coding

< Suclg2-201protein coding

< Suclg2-202lncRNA < Suclg2-203retained intron

< Suclg2-205protein coding

Regulatory Build

95.5Mb 95.6Mb 95.7Mb Reverse strand 265.79 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000204224

< Suclg2-204protein coding

Reverse strand 245.79 kb

ENSMUSP00000144... Low complexity (Seg) TIGRFAM Succinate--CoA synthetase, beta subunit Superfamily SSF56059 Succinyl-CoA synthetase-like

Pfam ATP-grasp fold, succinyl-CoA synthetase-type ATP-citrate lyase/succinyl-CoA ligase

PROSITE profiles ATP-grasp fold PROSITE patterns Succinyl-CoA synthetase, beta subunit, conserved site PIRSF Succinate--CoA synthetase, beta subunit PANTHER Succinate--CoA synthetase, beta subunit

PTHR11815:SF11 HAMAP Succinate--CoA synthetase, beta subunit

Succinate--CoA ligase [GDP-forming] subunit beta, mitochondrial Gene3D 3.30.470.20 Succinyl-CoA synthetase-like

ATP-grasp fold, subdomain 1

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 433

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

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