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Mouse Ccnl2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ccnl2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ccnl2 gene (NCBI Reference Sequence: NM_207678 ; Ensembl: ENSMUSG00000029068 ) is located on Mouse chromosome 4. 11 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000030944). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ccnl2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-128M14 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 5 starts from about 37.9% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2559 bp, and the size of intron 5 for 3'-loxP site insertion: 2341 bp. The size of effective cKO region: ~565 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 5 11 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ccnl2 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(7065bp) | A(25.14% 1776) | C(20.3% 1434) | T(32.29% 2281) | G(22.28% 1574)

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% chr4 + 155814674 155817673 3000 browser details YourSeq 183 1060 1371 3000 93.0% chr1 + 157420933 157421408 476 browser details YourSeq 170 1060 1415 3000 87.0% chr4 + 59056491 59056745 255 browser details YourSeq 157 1218 1417 3000 96.5% chr4 - 125980535 125980862 328 browser details YourSeq 148 2011 2334 3000 89.8% chr4 - 129277829 129278160 332 browser details YourSeq 147 1218 1749 3000 83.1% chr5 - 88681912 88682199 288 browser details YourSeq 144 1117 1372 3000 93.9% chr4 - 59799680 59800019 340 browser details YourSeq 139 1218 1421 3000 87.7% chr11 - 23466118 23466283 166 browser details YourSeq 139 1216 1416 3000 87.5% chr18 + 30661921 30662091 171 browser details YourSeq 136 1210 1372 3000 90.0% chr5 - 97553791 97553941 151 browser details YourSeq 135 1218 1372 3000 91.7% chr2 - 48034009 48034152 144 browser details YourSeq 134 1218 1372 3000 91.6% chr10 - 110791040 110791182 143 browser details YourSeq 134 2010 2415 3000 86.5% chr1 - 86050154 86050691 538 browser details YourSeq 133 1218 1395 3000 89.3% chr12 - 73971049 73971188 140 browser details YourSeq 132 1210 1370 3000 89.8% chr13 - 67676823 67676970 148 browser details YourSeq 132 1218 1372 3000 91.1% chr11 - 107183042 107183188 147 browser details YourSeq 131 1218 1409 3000 86.5% chr17 - 9257618 9257768 151 browser details YourSeq 131 1216 1372 3000 91.0% chr1 + 3385056 3385202 147 browser details YourSeq 128 1216 1372 3000 92.8% chr10 + 56425956 56426107 152 browser details YourSeq 128 1218 1418 3000 86.4% chr1 + 178257376 178257532 157

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% chr4 + 155818239 155821238 3000 browser details YourSeq 68 2928 3000 3000 93.0% chr5 - 77224010 77224080 71 browser details YourSeq 66 2929 3000 3000 97.2% chr7 + 99310784 99310865 82 browser details YourSeq 65 2923 3000 3000 94.5% chr13 - 62696378 62696462 85 browser details YourSeq 63 2928 3000 3000 97.1% chr11 - 5537545 5537619 75 browser details YourSeq 62 2936 3000 3000 98.5% chr2 - 70518320 70518385 66 browser details YourSeq 62 2932 3000 3000 98.5% chr2 + 5029107 5029177 71 browser details YourSeq 61 2926 3000 3000 97.1% chr9 + 53778142 53778220 79 browser details YourSeq 61 2935 3000 3000 97.0% chr1 + 167452831 167452897 67 browser details YourSeq 60 2930 3000 3000 92.9% chr9 - 70637471 70637548 78 browser details YourSeq 60 2931 3000 3000 98.4% chr15 + 79894210 79894279 70 browser details YourSeq 59 2931 3000 3000 96.9% chr2 + 34792765 34792835 71 browser details YourSeq 58 2928 3000 3000 98.4% chr19 - 33404721 33404793 73 browser details YourSeq 58 2928 3000 3000 95.4% chr10 + 4393738 4393811 74 browser details YourSeq 57 2928 3000 3000 98.4% chr9 - 62326546 62326620 75 browser details YourSeq 57 2929 3000 3000 93.9% chr15 - 41086641 41086714 74 browser details YourSeq 56 2936 3000 3000 98.3% chr14 - 28360627 28360692 66 browser details YourSeq 55 2934 3000 3000 95.1% chr6 + 149321173 149321241 69 browser details YourSeq 55 2935 3000 3000 96.7% chr5 + 96540051 96540117 67 browser details YourSeq 55 2935 3000 3000 93.0% chr2 + 59638031 59638094 64

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ccnl2 cyclin L2 [ Mus musculus (house mouse) ] Gene ID: 56036, updated on 12-Aug-2019

Gene summary

Official Symbol Ccnl2 provided by MGI Official Full Name cyclin L2 provided by MGI Primary source MGI:MGI:1927119 See related Ensembl:ENSMUSG00000029068 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 Pcee; SB138; Ania-6b; AA409936; AW261738; HCLA-150; 1700010A01Rik; 1810019L15Rik; 2010319M22Rik Expression Ubiquitous expression in limb E14.5 (RPKM 65.5), bladder adult (RPKM 45.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 4 E2; 4 87.47 cM See Ccnl2 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (155810219..155824543)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (155186598..155198652)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 5 transcripts

Gene: Ccnl2 ENSMUSG00000029068

Description cyclin L2 [Source:MGI Symbol;Acc:MGI:1927119] Gene Synonyms 1700010A01Rik, 1810019L15Rik, 2010319M22Rik, Pcee, ania-6b Location Chromosome 4: 155,812,489-155,824,543 forward strand. GRCm38:CM000997.2 About this gene This gene has 5 transcripts (splice variants), 162 orthologues, 6 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ccnl2- ENSMUST00000030944.10 2442 518aa ENSMUSP00000030944.4 Protein coding CCDS19042 Q9JJA7 TSL:1 201 GENCODE basic APPRIS P1

Ccnl2- ENSMUST00000126346.1 1894 93aa ENSMUSP00000116000.1 Nonsense mediated - F6RXL0 CDS 5' 202 decay incomplete TSL:5

Ccnl2- ENSMUST00000139066.7 4220 No - Retained intron - - TSL:1 205 protein

Ccnl2- ENSMUST00000129850.7 3411 No - Retained intron - - TSL:1 203 protein

Ccnl2- ENSMUST00000136370.1 833 No - Retained intron - - TSL:1 204 protein

32.05 kb Forward strand 155.81Mb 155.82Mb 155.83Mb Genes (Comprehensive set... Mrpl20-205 >lncRNA Ccnl2-201 >protein coding Aurkaip1-206 >retained intron

Mrpl20-201 >protein coding Ccnl2-205 >retained intron Aurkaip1-205 >protein coding

Mrpl20-206 >protein coding Ccnl2-203 >retained intron Aurkaip1-201 >protein coding

Mrpl20-203 >nonsense mediated decay Ccnl2-204 >retained intron Aurkaip1-203 >protein coding

Mrpl20-202 >protein coding Ccnl2-202 >nonsense mediated decay Aurkaip1-204 >lncRNA

Mrpl20-204 >lncRNA Aurkaip1-202 >protein coding

Contigs AL670236.9 > Regulatory Build

155.81Mb 155.82Mb 155.83Mb Reverse strand 32.05 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000030944

12.05 kb Forward strand

Ccnl2-201 >protein coding

protein_pic

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

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