In 2014, researchers combined DNA barcoding with next-generation sequencing (NGS) to develop the adeno-associated virus (AAV) barcode-seq technology for streamlining AAV screening. Using this method, scientists can directly compare the performance of different AAV simultaneously in a single animal.
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(1) To track the expression of a specific AAV genome in a pool, researchers add a unique pair of small barcodes consisting of a few nucleotides to the AAV sequence. Each viral genome also contains two flanking inverted terminal repeats (ITR) that enclose the following genes: a rep gene, which is required for viral genome replication and packaging; a cap gene, which is required for the expression of capsid proteins; and a polyadenylation signal (pA), which ensures proper processing and stability of the mRNA product. (2) Scientists then mix the different barcoded AAV to form an AAV pool or library. (3) Researchers inject the AAV mixture into a living model, such as a mouse. The different AAV will transduce, or infect, the animals’ tissues. (4) Scientists collect the tissues of interest and extract DNA from the different cells. (5) Viral DNA from the different AAV is amplified and prepared for NGS. (6) Researchers analyze NGS data to study patterns of viral DNA expression, which may identify AAV that have a trophism for certain tissues, and may thus serve as better gene delivery vehicles when targeting that specific tissue. |
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