Guide-itTM Genotype Confirmation Kit

Description

The CRISPR/Cas9 system has been harnessed to create a simple, RNA-programmable method to mediate genome editing in mammalian cells. CRISPR/Cas9 editing generates insertions or deletions indels that can result in gene knockout. In most cases, cells have two copies of any given gene and indel mutations can be generated in either one or both alleles. The Guide-it Genotype Confirmation Kit provides a simple protocol to determine whether gene editing resulted in indels on one allele monoallelic or both alleles biallelic in singly isolated cells clones , allowing for the identification of clones with desired mutations for further analysis. The kit is designed to be used in conjunction with the Guide-it sgRNAIn VitroTranscription Kit Cat. No. 632635 , which generates a target-specific guide RNA used in the genotype confirmation reaction. The sequence of the guide RNA used for the genotype confirmation reaction is identical to the guide RNA used for the initial genome editing experiment. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells.The CRISPR/Cas9 system relies on a single guide RNA sgRNA directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1 nonhomologous end joining, NHEJ resulting in gene knockout due to error-prone repair orange , or 2 homology-directed repair HDR , resulting in gene knockin due to the presence of a homologous repair template purple . Back Schematic of the Guide-it Genotype Confirmation Kit workflow.Single-cell clones are generated from mammalian cells treated with Cas9 and a gene-specific single guide RNA sgRNA . Crude extracts are prepared from the clones, and the target gene is PCR amplified. The amplification product is then used in anin vitrocleavage reaction with Cas9 and the same sgRNA that was used for genome editing in cells. The cleavage reaction is then run on an agarose gel. The presence of any indel in the target gene would preclude cleavage of the amplicon by Cas9, while wild-type DNA that does not have indels would be cleaved. A monoallelic genotype can be distinguished from a biallelic genotype by the presence of both cut and uncut DNA bands. Back Accurate determination of genotype in HEK 293 cells.Panel A. HEK 293 cells were treated with Cas9 and a sgRNA targeting theC4BPBgene. Fifteen single-cell clones were generated, and the Guide-it Genotype Confirmation Kit was used to determine the genotype at theC4BPBlocus. Wild-type WT , monoaleic M , and biallelic B control reactions were included in the analysis left panel . The results indicated that the clones 1, 4, 10, and 12 are wild-type; clone 2 is monoallelic; and clones 3, 59, 11 and 1315 are biallelic.Panel B.Sequencing results for select clones from panel A. For each result, the lowercase letters represent the WT sequence. For clones identified as biallelic in the genotype confirmation assay Panel A , sequencing indicated that clone 7 is heterozygous and clone 9 and 11 are homozygous. In clone 2, three different alleles were detected; it is possible that this result stems from copy number variation in the HEK 293 cell line. Back Genotype determination byin vitroCas9/sgRNA cleavage.After amplification of the target region, amplicons are used in a Cas9/sgRNA-mdiatedin vitrocleavage reaction. In the case of a wild-type genotype WT , both alleles will be cleaved by the Cas9/sgRNA complex resulting in two small bands when the cleavage reaction is run on an agarose gel. However, for mutant cells, different banding patterns will be present depending on the genotype. For monoallelic mutants, only the amplified WT allele would be cleaved, resulting in two small bands and one large uncut band. For biallelic mutants, neither amplified allele would be cleaved, resulting in a single large band on the gel. Back