FIG 10.

Illustration of the effect of PAM position in the PBS DNA on Cas9/sgRNA restriction of HIV-1 infection. If PAM is positioned on the plus strand of the PBS, HIV-1 is able to escape by acquiring and maintaining single- or double-nucleotide mutations in the PBS. If PAM is located on the minus strand of the PBS DNA, Cas9/sgRNA is able to target and cleave viral cDNA that is reverse transcribed from viral RNA that bears mutated PBS sequences. Based on these mechanisms, it can be predicted that a greater and more persistent suppression of HIV-1 infection can be achieved with a Cas9 variant using the CCA PAM that is on the minus strand of the PBS DNA and also allows the design of sgRNA with its seed region (14 to 15 nt) included in the PBS.