RESUMEN
Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Técnicas de Sustitución del Gen , Transgenes/genéticaRESUMEN
We describe CALITAS, a CRISPR-Cas-aware aligner and integrated off-target search algorithm. CALITAS uses a modified and CRISPR-tuned version of the Needleman-Wunsch algorithm. It supports an unlimited number of mismatches and gaps and allows protospacer adjacent motif (PAM) mismatches or PAMless searches. CALITAS also includes an exhaustive search routine to scan genomes and genome variants provided with a standard Variant Call Format file. By default, CALITAS returns a single best alignment for a given off-target site, which is a significant improvement compared to other off-target algorithms, and it enables off-targets to be referenced directly using alignment coordinates. We validate and compare CALITAS using a selected set of target sites, as well as experimentally derived specificity data sets. In summary, CALITAS is a new tool for precise and relevant alignments and identification of candidate off-target sites across a genome. We believe it is the state of the art for CRISPR-Cas specificity assessments.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Simulación por Computador , Endonucleasas/genética , Algoritmos , Proteínas Bacterianas , Proteína 9 Asociada a CRISPR , Proteínas Asociadas a CRISPR , Endodesoxirribonucleasas , Edición Génica , Genoma , ARN Guía de Kinetoplastida/genética , Programas InformáticosRESUMEN
SUMMARY: High-throughput screening of the host transcriptional response to various viral infections provides a wealth of data, but utilization of microarray and next generation sequencing (NGS) data for analysis can be difficult. The Host Transcriptional Response DataBase (HoTResDB), allows visitors to access already processed microarray and NGS data from non-human primate models of viral hemorrhagic fever to better understand the host transcriptional response. AVAILABILITY: HoTResDB is freely available at http://hotresdb.bu.edu.