Published: March 31st, 2022
Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.
Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.
Maintaining genomic stability is critical for all living organisms. Accurate DNA replication and a robust DNA damage response (DDR) are necessary to faithfully propagate the genetic material1,2. DNA damages occur regularly in most cells2,3. When these damages are sensed, cell cycle progression is halted, and DNA repair mechanisms are activated. Double strand breaks in DNA or DSBs are the most toxic and mutagenic type of DNA damage3,4.
While several DDR signaling path....
1. Cell plating
Three representative results are presented for this protocol. Figure 1 is an immunoblot confirming expression of CAS9 in HFK control (LXSN) and HFK expressing beta-HPV 8E6 (8E6). 48 h after transfection, whole cell lysates were harvested and subsequently probed with an anti-CAS9 antibody (or GAPDH as a loading control). The result shows that HFK LXSN and HFK 8E6 are expressing similar amount of CAS9 indicating that transfection efficiency is similar between t.......
In addition to the depth of information provided, there are several advantages to this method. First, DSB repair, in theory, can be assessed at any genomic loci without modifying the genome of the cell of interest. Second, access to NGS analysis of repair is increased by the reduced cost and computational effort afforded by making and analyzing a single DSB targeted to a defined area. Finally, with the genomes of additional organisms routinely becoming available and multiple publications demonstrating successful transfec.......
Research reported in this manuscript was supported by the National Institute of General Medical Sciences of the National Institutes of Health (P20GM130448) (NAW and RP); National Cancer Institute of the National Institutes of Health (NCI R15 CA242057 01A1); Johnson Cancer Research Center in Kansas State University; and the U.S. Department of Defense (CMDRP PRCRP CA160224 (NAW)). We appreciate KSU-CVM Confocal Core and Joel Sanneman for our immunofluorescence microscopy. The content is solely the responsibility of the authors and does not necessarily represent the official views of these funding agencies.....
|6 Well Tissue Culture Plate
|Cell culture plate
|BCA assay kit
|Centrifuge 5910 R
|CLC Genomic Workbench
|deep sequence data analysis software/indel caller/variant caller
|Digital Microplate Genie pulse
|DYKDDDDK Tag Monoclonal Antibody (FG4R)
|Epilife CF Kit
|Cell cultrue media and supplements
|Fetal Bovine Serum (FBS)
|Cell culture supplement
|Goat anti-Rabbit IgG
|HighPrep PCR Clean-up system
|Bead-based PCR cleanup kit
|KAPA HiFi HotStart ReadyMix PCR Kit
|PCR mastermix/PCR assay
|MagAttract HMW DNA kit
|High Molecular Weight DNA extraction kit
|Thermo Fisher Scientific
|96-Well Magnetic Rack
|MiniAmp Thermal Cycler
|Miseq v2 300 cycle reagent kit
|300-cycle cartridge/sequencing reagents
|Nextera XT DNA Library Prep kit
|Library preparation kit
|Nextera XT Kit v2 Set A
|Nunc 96-well polypropylene DeepWell Stroage plates
|Thermo Fisher Scientific
|deep well 96-well plates
|Penicillin-Streptomycin Solution (100X)
|Antibiotics for cell culture
|Phosphate Buffered Saline (PBS)
|SgRNA/CAS9 plasmids targeting 5’- GGCGTATCTGTGTGAGGACT
|QIAxcel Advanced System
|capillary electrophersis machine
|QIAxcel DNA screening kit
|DNA buffer/ capillary electrophersis tubes
|Qubit 1x ds HS Assay Kit
|Fluorometer reagents/1x dsDNA solution
|Qubit 4 Fluorometer
|Qubit Assay Tubes
|Thermo Fisher Scientific
|Fluorometer assay tubes
|RIPA Lysis Buffer
|Lysis buffer for protein extraction
|Trypsin-EDTA (0.05%), phenol red
|Xfect Transfection Reagent
|genomic data analysis software
|CLC Workbench v21.0.
|Data analysis software
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