# Nano-Polymers as Cas9 Inhibitors
## 4 Discussion  
The repertoire of CRSIPR-Cas inhibitors has been rapidly expanding with new tools available to search for and identify these molecules. By origin, the Cas9 inhibitors can be divided into two groups: (1) naturally occurring Cas9 inhibitors, such as the Acr proteins; and (2) engineered Cas9 inhibitors. Recently, a machine-learning-based approach predicted over 2500 candidate anti-CRISPR (Acr) families in phage [30], indicating the existence of a gigantic number of Acr proteins in nature. More than 80 distinct Acr families have been identified to date that inhibit type I, II, III, V, and VI CRISPR-Cas systems [31], among which AcrIIA and AcIIC are Cas9-specific. Acr families have diverse modes of interaction with Cas proteins. As for the type-II Acr proteins, their inhibition mechanisms include the following: (1) inhibition of sgRNA binding (e.g., AcrIIA17 and AcrIIC2) [32, 33]; (2) blocking target DNA entry by occupying the PAM binding site (e.g., AcrIIA2, AcrIIA4 and AcrIIC5) [22, 29, 34, 35]; (3) interacting with the Cas9 catalytic domain (e.g., AcrIIC1 and AcrIIC3) [36, 37, 38] to inhibit its function; and (4) other unclear CRISPR inhibitory mechanisms, such as a dimerized AcrIIA1 with a nuclear acid binding affinity [39]. In addition to natural Acr proteins, researchers have engineered synthetic inhibitors to achieve more precise control over Cas9 activity. These synthetic inhibitors often aim to increase the specificity of genome editing or enable conditional activation or repression of Cas9. These engineered Cas9 inhibitors include both small molecules and peptide-based Cas9 inhibitors through rational design and screening [40, 41]. To the best of our knowledge, our current work is the first report of nano-polymer Cas9 inhibitors identified to date. More interestingly, the linear polymer backbone conjugated with the common trileucine peptide appeared to be a common structural basis for this new class of Cas9 inhibitors to block the endonuclease function of Cas9. The LLL moiety has been previously used as an efficient mechanism for nanodrug to escape endosomes and avoid degradation, and our current work has added a new layer of functional complexity to it in the potential regulation of Cas9 function. All five different nano-polymers we screened for potential Cas9 inhibition are over 50,000 Daltons in molecular weight, and three of them exhibited varying efficiency in blocking Cas9 nuclease function. The largest polymer, PMLA-LLL, displayed the strongest Cas9 inhibition with a minimal polymer/RNP molar ratio of 3:1 to completely inhibit Cas9-mediated DNA cleavage, followed by PGA200-LLL and PLD200-LLL, sequentially. The HPLC spectra provide robust evidence of the dose-dependent binding between PMLA-LLL and Cas9. The reduction in the free Cas9 peak and the progressive increase in the PMLA-LLL/Cas9 complex peak support the high binding efficiency of PMLA-LLL. This behavior underscores its capability to encapsulate and stabilize the Cas9 protein effectively. Although PGA200-LLL and PLD200-LLL are quite close in their overall size (Table 3), the two polymers showed a substantial difference in their efficiency as Cas9 inhibitors (Figure 3), suggesting that the polymer structural differences are the major determinants of the Cas9 inhibitory efficiency. Interestingly, with the same backbone structure, there appears to be a requirement of length of the polymers to block Cas9 function, as the shorter forms of PGA-LLL showed a decreased efficiency in Cas9 inhibition. This study also has several limitations. Firstly, although theoretical polymer-Cas9 binding models were generated computationally, the definitive structure of nano-polymer Cas9 inhibitor binding to the Cas9 protein still need to be established. Secondly, the stability of nano-polymer-LLL Cas9 inhibitors needs to be determined to better evaluate their potential in CRISPR/Cas9 functional regulation.