https://www.alphaknockout.com

Mouse Bcl2l11 Knockout Project (CRISPR/Cas9)

Objective: To create a Bcl2l11 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Bcl2l11 gene (NCBI Reference Sequence: NM_207680 ; Ensembl: ENSMUSG00000027381 ) is located on Mouse chromosome 2. 4 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000110341). 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: Consistent with this gene's role in hematopoietic homeostasis, homozygous null mutants accumulate lymphoid and myeloid cells and succumb to autoimmune kidney disease.

Exon 2 starts from the coding region. Exon 2 covers 64.97% of the coding region. The size of effective KO region: ~395 bp. The KO 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 4

Legends Exon of mouse Bcl2l11 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. 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 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. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

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

Sequence 12

Summary: Full Length(2000bp) | A(19.1% 382) | C(27.8% 556) | T(20.45% 409) | G(32.65% 653)

Note: The 2000 bp section upstream of Exon 2 is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

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

Sequence 12

Summary: Full Length(2000bp) | A(28.15% 563) | C(18.4% 368) | T(31.8% 636) | G(21.65% 433)

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% chr2 + 128126634 128128633 2000 browser details YourSeq 34 1905 1969 2000 71.8% chr4 + 46015286 46015335 50 browser details YourSeq 24 1070 1106 2000 83.4% chr2 + 30733584 30733621 38 browser details YourSeq 22 1911 1932 2000 100.0% chr1 + 118734092 118734113 22 browser details YourSeq 21 1916 1940 2000 92.0% chr1 - 43421495 43421519 25 browser details YourSeq 20 1789 1808 2000 100.0% chr10 - 120483151 120483170 20 browser details YourSeq 20 1697 1716 2000 100.0% chr1 - 58213949 58213968 20 browser details YourSeq 20 1685 1704 2000 100.0% chr1 + 9568464 9568483 20

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% chr2 + 128129016 128131015 2000 browser details YourSeq 25 1756 1780 2000 100.0% chr17 + 58599904 58599928 25 browser details YourSeq 24 238 261 2000 100.0% chr17 - 43560390 43560413 24 browser details YourSeq 23 1977 1999 2000 100.0% chr4 + 97404279 97404301 23 browser details YourSeq 23 1119 1144 2000 96.0% chr12 + 10369305 10369332 28 browser details YourSeq 21 564 584 2000 100.0% chr1 - 76804296 76804316 21

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: Bcl2l11 BCL2-like 11 (apoptosis facilitator) [ Mus musculus (house mouse) ] Gene ID: 12125, updated on 10-Oct-2019

Gene summary

Official Symbol Bcl2l11 provided by MGI Official Full Name BCL2-like 11 (apoptosis facilitator) provided by MGI Primary source MGI:MGI:1197519 See related Ensembl:ENSMUSG00000027381 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 Bim; Bod; bcl2-L-11; 1500006F24Rik Expression Ubiquitous expression in thymus adult (RPKM 7.8), lung adult (RPKM 4.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 F1 See Bcl2l11 in Genome Data Viewer Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (128126038..128162547)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (127951774..127988283)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 8 transcripts

Gene: Bcl2l11 ENSMUSG00000027381

Description BCL2-like 11 (apoptosis facilitator) [Source:MGI Symbol;Acc:MGI:1197519] Gene Synonyms 1500006F24Rik, Bim, Bod Location Chromosome 2: 128,126,038-128,162,547 forward strand. GRCm38:CM000995.2 About this gene This gene has 8 transcripts (splice variants), 103 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 74 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Bcl2l11- ENSMUST00000110341.8 5036 196aa ENSMUSP00000105970.2 Protein CCDS16713 O54918 Q542N5 TSL:1 205 coding GENCODE basic APPRIS P4

Bcl2l11- ENSMUST00000103210.1 3538 140aa ENSMUSP00000099499.1 Protein CCDS16714 O54918 TSL:1 203 coding GENCODE basic APPRIS ALT2

Bcl2l11- ENSMUST00000019281.13 2490 110aa ENSMUSP00000019281.7 Protein CCDS16712 O54918 TSL:1 201 coding Q3U7X3 GENCODE basic APPRIS ALT2

Bcl2l11- ENSMUST00000089634.11 2301 91aa ENSMUSP00000087062.5 Protein CCDS71141 Q6PEB3 TSL:1 202 coding GENCODE basic

Bcl2l11- ENSMUST00000103211.7 672 110aa ENSMUSP00000099500.1 Protein CCDS16712 O54918 TSL:2 204 coding Q3U7X3 GENCODE basic APPRIS ALT2

Bcl2l11- ENSMUST00000146023.7 1611 No - lncRNA - - TSL:2 208 protein

Bcl2l11- ENSMUST00000129206.1 974 No - lncRNA - - TSL:1 206 protein

Bcl2l11- ENSMUST00000132201.1 391 No - lncRNA - - TSL:2 207 protein

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56.51 kb Forward strand 128.12Mb 128.13Mb 128.14Mb 128.15Mb 128.16Mb 128.17Mb Genes (Comprehensive set... Acoxl-201 >protein coding Bcl2l11-201 >protein coding

Acoxl-202 >protein coding Bcl2l11-202 >protein coding

Bcl2l11-208 >lncRNA

Bcl2l11-205 >protein coding

Bcl2l11-204 >protein coding

Bcl2l11-203 >protein coding

Bcl2l11-206 >lncRNA

Bcl2l11-207 >lncRNA

Contigs AL805950.11 > Regulatory Build

128.12Mb 128.13Mb 128.14Mb 128.15Mb 128.16Mb 128.17Mb Reverse strand 56.51 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000110341

36.51 kb Forward strand

Bcl2l11-205 >protein coding

ENSMUSP00000105... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Pfam Apoptosis, Bim N-terminal Bcl-x interacting, BH3 domain

PIRSF Bcl-2-like protein 11

PANTHER PTHR12044:SF9

PTHR12044

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

Variant Legend synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 196

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

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