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CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing [10] was the AAAS's choice for Breakthrough of the Year in 2015. [11] Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos. [12]
Designer nuclease systems such as CRISPR-cas9 are becoming increasingly popular research tools as a result of their simplicity, scalability and affordability. [10] [11] With this being said, off-target genetic modifications are frequent and can alter the function of otherwise intact genes. Multiple studies using early CRISPR-cas9 agents found ...
Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a
Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and open up specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within living organisms.
The PAM is required for a Cas nuclease to cut and is usually located 3-4 nucleotides downstream from the cut site. Once the gRNA base pairs with the target, Cas9 induces a double-strand break about 3 nucleotides upstream of the PAM. [27] [28] The optimal GC content of the guide sequence should be over 50%.
As CRISPR continues to exhibit low noise and minimal off-target effects, an alternative strategy is to reduce the number of sgRNAs per gene for a primary screen. Less stringent cut-offs are used for hit selection, and additional sgRNAs are later used in a more specific secondary screen. This approach is demonstrated by Doench et al.
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