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<p>After the workshop conducted at one-north festival, we requested the public to participate in a survey. This survey was designed to gauge public interest and reservations about the use of Cas9 for therapeutic applications.</p> | <p>After the workshop conducted at one-north festival, we requested the public to participate in a survey. This survey was designed to gauge public interest and reservations about the use of Cas9 for therapeutic applications.</p> | ||
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<p>An overwhelmingly high number (85%) of participants is receptive to using a fully mature CRISPR/Cas9 technology – that is without any side effects. </p> | <p>An overwhelmingly high number (85%) of participants is receptive to using a fully mature CRISPR/Cas9 technology – that is without any side effects. </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/5/5a/Ntu_ihp_survey2.png" > | + | <img src="https://static.igem.org/mediawiki/2017/5/5a/Ntu_ihp_survey2.png" width="500" height="400"> |
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<p>When asked to consider whether they would be receptive to using CRISPR/Cas9 technology in the event of personal suffering to lung cancer – even when the issues of the treatment have yet to be fully worked out, more than half stated that they would, while 30% would consider the treatment. </p> | <p>When asked to consider whether they would be receptive to using CRISPR/Cas9 technology in the event of personal suffering to lung cancer – even when the issues of the treatment have yet to be fully worked out, more than half stated that they would, while 30% would consider the treatment. </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/b/bd/Ntu_ihp_survey3.png" > | + | <img src="https://static.igem.org/mediawiki/2017/b/bd/Ntu_ihp_survey3.png" width="500" height="400"> |
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<p>The T7E1 results for A549 cells with 24h transfection time were all negative as we expected, suggesting that CRISPR-Cas could not target the wild-type EGFR gene with all the sgRNAs designed. Surprisingly, positive T7E1 results were observed for some sgRNAs when A549 cells were transfected with 48h time, which may indicate that CRISPR-Cas could possibly tolerate the mutation and perform cleavage. Another parameter which could matter is the transfection time, since the cuts were only observed after 48h transfection time but not in 24h transfection time. This could mean that longer transfection time may affect the accuracy of CRISPR-Cas as well.</p> | <p>The T7E1 results for A549 cells with 24h transfection time were all negative as we expected, suggesting that CRISPR-Cas could not target the wild-type EGFR gene with all the sgRNAs designed. Surprisingly, positive T7E1 results were observed for some sgRNAs when A549 cells were transfected with 48h time, which may indicate that CRISPR-Cas could possibly tolerate the mutation and perform cleavage. Another parameter which could matter is the transfection time, since the cuts were only observed after 48h transfection time but not in 24h transfection time. This could mean that longer transfection time may affect the accuracy of CRISPR-Cas as well.</p> | ||
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+ | <p>However, we cannot be confident enough to conclude that this indicates the off-target effect of CRISPR-Cas, since there were no duplicates generated due to time constraint. Besides carrying out duplicates, the confirmation of off-targets can be carried out using various techniques such as using in-vitro cutting assay or using different cell lines such as primary cell lines and HEK293 cells.</p> | ||
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+ | <p>Over-generalized conclusion is a concern in scientific research as it may lead to the generation of insignificant results. An example of such a publication will be the paper titled, “Unexpected mutations after CRISPR-Cas9 editing in-vivo”.</p> | ||
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+ | <p>In the paper, the authors conclude that CRISPR-Cas9 editing in blind mice that is supposed to only correct a mutation in the Pde6b gene, also causes a large number of mutations in untargeted regions. It is because they found an unexpectedly high number of indels and single nucleotide polymorphisms (SNPs) in the CRISPR-treated mice. This conclusion is based on the assumption that the 3 mice (1 control mouse and 2 CRISPR-Cas9 treated mice) used as the subjects of the experiment were genetically identical prior to the treatment. </p> | ||
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+ | <h3>Data Analysis Methodology</h3> | ||
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+ | <p>To validate if the claims are really true we attempted to reidentify the SNPs and indels by using Genome Analysis ToolKit (GATK) best practices. Three mice are used as the subjects of the experiment (1 control mouse: FVB and 2 CRISPR-Cas9 treated mice: F03 and F05). Firstly, we realigned the raw sequencing data obtained from SRR5450996 (FVB control mouse), SRR5450997 (F03), SRR5450998 (F05) with mouse reference genome, mm10, by using Burrows-Wheeler Aligner (bwa mem), resulting in a BAM file. The duplicates are then marked and removed using MarkDuplicates tool from Picard. The following steps, AddOrReplaceReadGroups. Information like read group ID, read group library, read group platform, and sample name are added. This step is added because it is required by GATK pipeline for SNP and indel calling in later steps. SNP calling is locating SNPs in comparison of reference genome while indel detects the insertions and deletions with respect to the reference genome. After this step, the sequencing is now ready to be fed into GATK pipeline.</p> | ||
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+ | <p>Firstly, the BAM files are realigned to produce better local alignments by using RealignerTargetCreator, IndelRealigner, Base recalibrator and PrintReads to create the final BAM file. SNPs and indels are called by using HaplotypeCaller, with mouse dbSNP142 as the reference database. The resulting output is a vcf file containing the location of indels and SNPs. SNPs are extracted from the callsets by setting SNP as the selection type for SelectVariants. The subsequent step is to apply a filter to the SNPs, the filtering is done by using hard filtering with the parameters recommended by GATK.</p> | ||
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+ | <p>There are 61034 numbers of shared SNPs between F03 and F05 mice, that are not found in FVB mouse, and therefore are unique SNPs between F03 and F05 mice. In addition, there are 10837 SNPs between FVB and F05 mouse, and only 9236 number of SNPs shared between FVB and F03 mouse. The bar graphs below show the distribution of number of SNPs across different chromosomes.</p> | ||
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<p>Over-generalized conclusion is a concern in scientific research as it may lead to the generation of insignificant results. An example of such a publication will be the paper titled, “Unexpected mutations after CRISPR-Cas9 editing in-vivo”.</p> | <p>Over-generalized conclusion is a concern in scientific research as it may lead to the generation of insignificant results. An example of such a publication will be the paper titled, “Unexpected mutations after CRISPR-Cas9 editing in-vivo”.</p> | ||
<p>In the paper, the authors conclude that CRISPR-Cas9 editing in blind mice that is supposed to only correct a mutation in the Pde6b gene, also causes a large number of mutations in untargeted regions. It is because they found an unexpectedly high number of indels and single nucleotide polymorphisms (SNPs) in the CRISPR-treated mice. This conclusion is based on the assumption that the 3 mice (1 control mouse and 2 CRISPR-Cas9 treated mice) used as the subjects of the experiment were genetically identical prior to the treatment. Since then, a number of articles has been published to rebut this claim and the paper had received a second editorial.</p> | <p>In the paper, the authors conclude that CRISPR-Cas9 editing in blind mice that is supposed to only correct a mutation in the Pde6b gene, also causes a large number of mutations in untargeted regions. It is because they found an unexpectedly high number of indels and single nucleotide polymorphisms (SNPs) in the CRISPR-treated mice. This conclusion is based on the assumption that the 3 mice (1 control mouse and 2 CRISPR-Cas9 treated mice) used as the subjects of the experiment were genetically identical prior to the treatment. Since then, a number of articles has been published to rebut this claim and the paper had received a second editorial.</p> | ||
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Revision as of 18:32, 1 November 2017