RESUMEN
The programmable sequence specificity of CRISPR has found uses in gene editing and diagnostics. This manuscript describes an additional application of CRISPR through a family of novel DNA enrichment technologies. CAMP (CRISPR Associated Multiplexed PCR) and cCAMP (chimeric CRISPR Associated Multiplexed PCR) utilize the sequence specificity of the Cas9/sgRNA complex to target loci for the ligation of a universal adapter that is used for subsequent amplification. cTRACE (chimeric Targeting Rare Alleles with CRISPR-based Enrichment) also applies this method to use Cas9/sgRNA to target loci for the addition of universal adapters, however it has an additional selection for specific mutations through the use of an allele-specific primer. These three methods can produce multiplex PCR that significantly reduces the optimization required for every target. The methods are also not specific to any downstream analytical platform. We additionally will present a mutation specific enrichment technology that is non-amplification based and leaves the DNA in its native state: TRACE (Targeting Rare Alleles with CRISPR-based Enrichment). TRACE utilizes the Cas9/sgRNA complex to sterically protect the ends of targeted sequences from exonuclease activity which digests both the normal variant as well as any off-target sequences.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica/métodos , Sitios Genéticos/genética , Polimorfismo de Nucleótido Simple/genética , Secuencia de Bases , Genoma Humano/genética , HumanosRESUMEN
Paper-based scaffolds support the three-dimensional culture of mammalian cells in tissue-like environments. These Tissue Papers, a name that highlights the use of materials obtained from (plant) tissue to generate newly functioning (human) tissue structures, are a promising analytical tool to quantify cellular responses in physiologically relevant extracellular gradients and coculture architectures. Here, we highlight current examples of Tissue Papers, commonly used methods of analysis, and current measurement challenges.