Question

3) Suggest three major potential reasons for off-target effects using the CRISPR/CAS system in gene editing.

Answer 1

Three major reasons for off target effects using CRISPR/CAS system in gene editing

1.Generation oF SNVs( Single nucleotide variants), the probable reason may be due to targeting of certain locii by the guide RNAs independent of their targets.

2.PAM sequence also affects the activity of sgRNA NGG ( N CAN BE A,T,C) is the canonical PAM sequence and NRG is the non canonical PAM sequence . The binding frequency of each nucleotide of the PAM SEQUENCE is different , thus NRG sequence is not optimal PAM sequence . This NRG and NGG sequence is not optimal for designing sgRNA.

3. Off target effects are also dependent on the type of cell we use. The integrity of double stranded DNA repair pathway is different in different cell types. e.g. Transformed cell have altered DSB/dysregulated cell lines (human)repair pathway whereas Pleuripotent Stem cell clones have intact DSB pathway and hence less off target effects can be seen in pleuripotent stem cell clones.

4 the seed sequence as well as PAM sequence must be designed carefully since seed sequences determines the binding via multiple ways or direct delivery of purified cas9 can be used to reduce off target effects.