HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

Our pooled sgRNA called the library screening, is a powerful genetic approach to identify and evaluate the biological function of the noncoding elements throughout the genome. This technique allows us to examine the effect of disrupting the binding sites associated, chromatic boundaries or other regulatory elements on targeted genes expression. Our approach can be used to identify the role of CTCF, long non-coding RNAs and enhancers, in HOX gene regulation in early embryonic development, as well as certain leukemias with an aberrant HOX gene signature.

Begin this procedure with the design of an sgRNA targeting CTCF binding sites. Cloning of the sgRNA library in cell preparation as described in the text protocol. To package the lentivirus, cotransfect HEK293 T cells with 20 micrograms of purified library vectors, 15 micrograms of the packaged plasmid, and 10 micrograms of the envelope plasmid, for 48 hours before harvesting the viruses.

After 48 hours, collect the virus supernatant and filter it through a 0.45 micron low-protein binding PVDF membrane. Next, concentrate the lentiviral supernatant by 50 fold, using the concentrator. Aliquot the concentrated viruses and store them in a minus 80 degrees celsius freezer.

Work in separate wells of a 12 well plate to mix MOLM13 cells with various doses of the concentrated lentivirus for six total groups. Immediately centrifuge these mixtures at 1000 times g for two hours at 33 degrees celsius. Transfer the 12 well plates back to the incubator at 37 degrees celsius and 5%carbon dioxide for four hours.

Gently aspirate the supernatant without disturbing the cell palette, and re-suspend the transduced cells with fresh supplemented RPMI 1640 Medium. Then transfer the re-suspended cells to T25 flasks and incubate at 37 degrees celsius for 48 hours without puromycin. After 48 hours split these cells into two flasks, an experimental group treated with one microliter per milliliter puromycin for five days, and a control group without puromycin treatment for five days.

Measure the optimized MOI value for transduction by dividing the number of live cells treated with puromycin by the number of cells without puromycin treatment. To transduce the cells with the pooled library, infect 1.5 million MOLM13 cells in a 6 well plate with 0.3 MOI of sgRNA pooled lentivirus in medium. Use the cells without the lentivirus infection as a control.

Immediately centrifuge the six well plate at 1000 times g for two hours at 33 degrees celsius to spinfect the cells. Then, transfer the plates back to the incubator at 37 degrees celsius in 5%carbon dioxide for four hours. Gently aspirate supernatant without disturbing the cell palette, and re-suspend the transduced cells with fresh medium.

Then, transfer the cells to T25 flasks and incubate at 37 degrees celsius for 48 hours without puromycin. After 48 hours, treat the cells with one microgram per milliliter puromycin for five days. Exchange for fresh medium after two days and maintain an optimal cell density.

Seed the single clone in 96 well plates with limiting dilution methods. Incubate these single clone at 37 degrees celsius and 5%carbon dioxide. Culture them for 3-4 weeks.

Count the sgRNA integrated MOLM13 cells with 0.4%Trypan Blue solution staining and then transfer 10, 000 live cells per well to a 96 well PCR plate. Centrifuge the plate at 1000 times g for five minutes and then thoroughly remove and discard the supernatant with a pipette. Avoid disturbing the cell palette.

After washing the cells as described in the text add 50 microliters of the cell lysis master mix to each well. Pipette up and down five times to re-suspend the cell palette. Incubate the mix for 10 minutes at room temperature.

Then, transfer the mixture to 37 degrees celsius for five minutes, and then 75 degrees celsius for an additional five minutes. Now add one microliter of cell lysate to the PCR wells containing the RT qPCR reaction mix. This mix includes the marker genes forward and reverse primer, reverse transcriptase and One-Step reaction mix.

Run the reverse transcription reaction for 10 minutes at 50 degrees celsius followed by polymerization activation, and DNA-denaturation for one minute at 95 degrees celsius. Perform RT PCR with 40 cycles of PCR reaction as detailed in the text protocol. Verify the HOXA9 decreased expression clones through Sanger sequencing and perform PCR with MOLM13 genome DNA as described in the text protocol.

Extract and purify the PCR products with the PCR purification kit. Ligate the purified PCR products into the T vector with T4 ligation buffer, T vector DNA, PCR DNA and T4 ligase. Place the ligation mix into an incubator at 16 degrees celsius overnight.

Transfer the ligation mix into DH5 alpha competent cells and plate on an LB ampicillin antibiotic agar plate. Incubate the plates overnight at 37 degrees celsius. Pick the single clones from the LB plate and verify them by genotyping and Sanger sequencing.

Set up the heteroduplex mixture group with 200 nanograms of the reference, and 200 nanograms of test PCR amplicons in a 0.2 milliliter PCR tube. Include the homoduplex mixture group with only 400 nanograms of reference PCR amplicons as a control. Separately incubate the heteroduplex and homoduplex mixture at 95 degrees celsius for five minutes in a one liter beaker filled with 800 milliliters of water.

Then, cool down the mixtures gradually to room temperature to anneal and form the heteroduplex or homoduplex. Now, separately digest 400 nanograms of the annealed heteroduplex and homoduplex mixture with one microliter of indel mutation detection nuclease, and two microliters of nuclease reaction buffer at 42 degrees celsius for 60 minutes. Analyze the digested samples with agarose gel electrophoresis.

The heteroduplex mixture DNA should be cut into small fragments and the homoduplex DNA should not be cut. sgRNA targeting MOLM13 positive clones were confirmed with the RT qPCR method, based on the expression levels of HOXA9 genes with the comparison with the control cells. Out of the 528 surviving clones screened, 10 clones exhibited more than 50%reduction in HOXA9 levels.

sgRNAs integrated into the HOXA9 reduced, HOXA9 unchanged and HOXA9 increased clones, were further confirmed by PCR amplification of the sgRNA and identification by Sanger sequence. Six of ten clones showing a reduction in HOXA9 levels contained sgRNAs targeting the CBS79 site, as determined by Sanger sequencing. Indel mutations of integrated sgRNA positive clones were determined by PCR-based genotyping and nuclease digestion.

The heterozygous deletion of the CTCF site located between HOXA7 and HOXA9 genes was identified by PCR-based genotyping. HOXA9-decreased clones five, six, 28 and 121 exhibited deletion in the CBS79 boundary location. Similar results were observed for the indel mutations in the CBS79 site that were analyzed by the nuclease digestion assay.

When performing this procedure, it's important to make equal amounts of heteroduplexes and homoduplexes in order to detect sgRNA-induced indel mutations through the nuclease digestion assay. Our approach enables utilization of the selected marker, to carry out next-generation sequencing on the target gene to discover the gene's effects on leukemogenesis. Overall our approach prevents RPC's for the functional correct and reduction of a noted genetic irregular elements during normal biological process and leukemogenesis in the posthumous genome project error.