Genome Engineering: Frontiers of CRISPR/Cas (CSHL)
Join Takara Bio at the Cold Spring Harbor Laboratory (CSHL) 2019 Genome Engineering meeting, scheduled for October 10–13. This meeting is specifically designed to foster creative interactions between researchers working on the basic biology of CRISPR/Cas and related bacterial defense systems and those applying this technology to a wide variety of organisms.
We're looking forward to seeing you at the meeting, where we will present a poster titled "Detecting allele-specific genome editing outcomes using a fluorescence-based screening method." For additional information, please check out our featured SNP screening kit and watch our recorded webinar about a fast and reliable method for SNP screening in genome editing experiments. Also, you can reach out to us with questions or requests via the "speak with us" link below.
CSHL Genome Engineering 2019: Poster
Detecting allele-specific genome editing outcomes using a fluorescence-based screening method
One of the most powerful applications of genome editing is the ability to introduce precise changes at genomic loci of interest. However, the success rate for these types of experiments is low since it relies on endogenous repair mechanisms. Therefore, optimization of the editing protocol, as well as the application of sensitive screening methods for identifying successfully edited clones, are essential factors for success.
To address this need, we developed a simple fluorescence-based method that enables detection of successful homology-directed repair (HDR) events independent of their length (from single-nucleotide substitutions to longer insertions) or the sequence at the targeted genomic site. The assay consists of PCR amplification of the genomic target site, followed by an enzymatic assay with a dual-color fluorescence-based readout using a standard plate reader. A positive fluorescent signal from the assay indicates the correct introduction of the desired edit.
Generating human disease models with heterozygous SNPs (one allele encoding the SNP, the other wild-type) is particularly challenging due to the low frequency of successful HDR and the propensity towards non-homologous end joining (NHEJ); as most edited clones encode the SNP in one allele and an indel in the other. Developed with these challenges in mind, the dual-color capability of our assay can be used to positively and unequivocally identify these rarely occurring heterozygous clones. As a test case, we introduced an SNP in the TIMP3 gene in human pluripotent stem cells (hiPSCs). To achieve a higher percentage of heterozygous clones, we used a mix of HDR templates encoding for the SNP or the wild-type allele, both with mutations at the PAM site to prevent re-cutting by the Cas9 endonuclease. With the help of the dual-color screening assay, heterozygous clones of interest were successfully identified and verified by Sanger sequencing.
Additionally, for scenarios involving knockin (KI) of longer sequences, the assay allows for the simultaneous detection of seamless insertions at both 5' and 3' ends of the recombinant sequence. As a test case, we engineered hiPSCs with a fusion of a myc tag with the UGT1A9 gene (related to drug metabolism) and screened the clonal cell lines with the dual-color screening assay. We were able to discern cell lines with either partial or complete insertions due to the assay's ability to interrogate both 5' and 3' ends of the insert.
CSHL Genome Engineering 2018: Poster
Streamlined production, application, and analysis of pooled genome-wide sgRNA lentiviral libraries
Genome-wide loss-of-function genetic screens are a powerful way to identify novel protein functions and biological processes within a cell. A common approach in in-vitro loss-of-function screens is to knock out genes in a population of cells, apply selective pressure, and then identify mutations that are either enriched or depleted in the selected population relative to a control. The easy programmability and high knockout efficiency of the CRISPR/Cas9 system has helped researchers maximize the potential of this in-vitro screening method to identify genes responsible for a given phenotype of interest. Current methods using pooled sgRNAs in loss-of-function screens rely on lentiviral vector-based delivery followed by next-generation sequencing (NGS) to analyze the resulting distribution of sgRNA sequences in screened cell populations. Inherent challenges include maintaining sgRNA representation in lentiviral plasmids, achieving optimal titers upon scale-up of lentivirus production, and preparing high-quality NGS libraries that accurately reflect the distribution of sgRNA sequences.
Here we present a streamlined approach for producing Cas9+/sgRNA+ cell populations in sufficient quantities for a genome-wide screen, and for generating NGS libraries used to assess changes in sgRNA representation, using the Guide-It CRISPR Genome-Wide sgRNA Library System. Our methods enable even novice users to perform genome-wide phenotypic screens without concerns for sgRNA representation, low virus titer, or NGS library preparation.
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