How to reduce off-target effects and increase CRISPR editing efficiency?

"Everyone is a writer" program aims to promote the sharing of knowledge and learning together. There are some answers about the CRISPR from this campaign.

Qian Zhang: As an emerging editing tool, CRISPR has attracted extensive attention in recent decades. However, its use has been largely limited due to its natural off-target effect. Therefore, in recent years, a large number of scientists have been trying to optimize the system in order to reduce off-target effects and increase editing efficiency. Mainly from the following aspects:

1. Improving sgRNA specificity

sgRNA near 10 ~ 12 bp base pairs is usually called PAM seed area, which determines the specificity of sgRNA and target DNA recognition. Learn More

Razeem MH: tools.However, potential off-target activities are crucial shortcomings in the CRISPR system. Researchers have proposed two types of off-target effects, the first types of off-target effects likely to occur due to the sequence homology of the target loci and the next types of off-target sites occur in the genome other than the target site.

Reduce off-target effects and increase CRISPR :

1)Guide RNA (gRNA) Modification and Engineering

a)    GC Content of sgRNA: The GC contents between 40%–60% in the gRNA sequence increase the on-target activity  because higher GC content stabilizes DNA: RNA duplex and destabilize the off-target binding.       Learn More

Elizabeth Gabriela Macedo Flores: Although various approaches have been shown to reveal the frequency of off-target mutations by at least an order of magnitude at several sites, it still remains unknown whether these RGEN variants are free of off-target effects in the entire genome. Recently, Digenome-seq, in vitro Cas9-digested whole-genome sequencing, has emerged as a robust, sensitive, unbiased, and cost-effective method for profiling genome-wide off-target effects of programmable nucleases, including Cas9, in human cells. Digenome-seq also consists of a two-stage process. Learn More

VINAY MEHRA: CRISPR has proven to be a highly versatile tool for gene editing with tremendous potential in a wide range of problems such as gene therapy, drug discovery, and genetic modification in plant technology. However, the accuracy and reliability of the CRISPR technology are severely hampered by the off-target effects, namely, the unintended cleavage of DNA at sites whose sequences show mismatches with the guide RNA (gRNA or sgRNA). Therefore, reducing the off-target effect becomes a timely critical issue in CRISPR genome editing technology. Learn More