Figure 1.

Gammaretrovirus-based Gag.MS2 chimera for CRISPR-Cas9 delivery

(A) Design of the gammaretroviral Gag.MS2 (g.Gag.MS2) variant and non-retroviral SpCas9.TS and sgRNA.TS expression plasmids. The configuration of structural Gag and enzymatic Pol proteins within gammaretroviral wt Gag-Pol (wt g.Gag-Pol) is depicted at the top. It consists of matrix (MA), p12, capsid (CA), nucleocapsid (NC), protease (PR), reverse transcriptase (RT), and integrase (IN) subdomains, which are separated from each other by individual protease sites. In g.Gag.MS2, the MS2CP dimer (2× MS2CP) protein replaces NC while maintaining the natural retroviral protease site (black bold bar). To ensure specific packaging of CRISPR-Cas9 RNA into assembling g.Gag.MS2 particles, two copies of MS2 target site (TS) are incorporated downstream of EGFP within the SpCas9.TS expression plasmid or were placed in (TS.inc) or adjacent (TS.adj) to the sgRNA scaffold in respective sgRNA expression plasmids. Co-expression of EGFP and DsRedexp helped to monitor transfection during particle production. (B) Direct comparison of integrating (g.RIT) and non-integrating g.Gag.MS2 CRISPR.Tet2 particles. Indicated supernatant volumes were used to transduce human HT1080-based RFP657.Tet2 reporter cells. Each data point represents one individually generated supernatant (n = 3). CMV, promoter from cytomegalovirus; hU6, human RNA Pol III U6 promoter; pA, poly(A) signal; PRE, post-transcriptional regulatory element from woodchuck hepatitis virus; SFFV, promoter from spleen focus forming virus.