Viral Vectors 101: Producing Your rAAV

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AAV infection of retinal neurons.
Overview of the parts of CRISPR. The bacterial chromosome encodes a tracrRNA (in some systems including Cas9), Cas proteins, and a CRISPR array. The CRISPR array is composed of identical repeat sequences and variable spacer sequences. The array is transcribed and processed into crRNAs, each including one repeat and one spacer. In bacteria, these crRNAs are bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs with the tracrRNA, and the spacer sequence is used to target complementary DNA sequences. In laboratory settings, an sgRNA includes the crRNA and tracrRNA sequences in a “single-guide RNA” that performs both functions. Cas9 cuts both the target and nontarget DNA strands upstream of the PAM site found in the nontarget strand.
Four different cells together. Three cells are each bound by an antibody with a red fluorescent probe. All three have arrows pointing to a trash can. The four cell is not bound by any antibody and has an arrow pointing to an open Eppendorf tube.
screenshot of various PRIDICT webpages
A cartoon schematic of prokaryotic chromosomal replication. The parent cell DNA is shown as a circular chromosome with a small region highlighted as the origin of replication, or ori.   During replication, the DNA helix has separated at the ori, creating a “bubble” of two single strands of DNA. The point of separation of the helix into these single strands is the replication fork. Two replication forks form on either end of the ori. At each replication fork, a helicase processively separates the DNA strands, and a polymerase synthesizes a new DNA strand paired to each single parent strand.  After termination, which is not shown in detail, the process results in two identical daughter cell DNA chromosomes.
addgenies smiling and talking together.

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