Evaluation of cas9 molecule/guide rna molecule complexes

Abstract

Disclosed herein are methods for evaluation, selection, optimization, and design of Cas9 molecule/gRNA molecule complexes.

1 - 57 . (canceled) 
     
     
         58 . A method of screening for a Cas9 molecule/gRNA molecule complex, the method comprising:
 (a) generating a plurality of samples, each sample comprising a Cas9 molecule/gRNA molecule complex generated by combining a Cas9 molecule and one of a plurality of gRNA molecules;   (b) detecting a melting temperature (T m ) value of the Cas9 molecule/gRNA molecule complex in each of the plurality of samples; and   (c) selecting at least one sample from the plurality of samples based on a comparison of the T m  values in the plurality of samples to a T m  value of a reference molecule or a T m  reference value.   
     
     
         59 . The method of  claim 58 , wherein the T m  value is detected using a thermal shift assay. 
     
     
         60 . The method of  claim 59 , wherein the thermal shift assay is selected from differential scanning fluorimetry (DSF), differential scanning calorimetry (DSC), or isothermal titration calorimetry (ITC). 
     
     
         61 . The method of  claim 60 , wherein the sample is selected if the T m  value of the Cas9 molecule/gRNA molecule complex in the sample is at least 1° C., at least 2° C., at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C., at least 10° C., at least 11° C., at least 12° C., at least 13° C., at least 14° C., at least 15° C., at least 16° C., at least 17° C., at least 18° C., at least 19° C., or at least 20° C. greater than the T m  value of the reference molecule or the T m  reference value. 
     
     
         62 . The method of  claim 61 , wherein the reference molecule is selected from the group consisting of:
 (a) a reference Cas9 molecule in the absence of a gRNA molecule;   (b) a reference Cas9 molecule complexed with a gRNA molecule different from the first gRNA molecule in the complex being evaluated; and   (c) a reference Cas9 molecule/gRNA molecule complex formed under a different condition or buffer than the Cas9 molecule/gRNA molecule complex being evaluated.   
     
     
         63 . The method of  claim 62 , wherein the condition comprises using a proportion of Cas9 molecule and gRNA molecule relative to the complex being evaluated. 
     
     
         64 . A method of determining the stability of a Cas9 molecule/gRNA molecule complex, the method comprising:
 (a) generating a plurality of Cas9 molecule/gRNA molecule complexes, each comprising a Cas9 molecule/gRNA molecule complex generated by combining a Cas9 molecule and one of a plurality of gRNA molecules;   (b) detecting a melting temperature (T m ) value of each of the Cas9 molecule/gRNA molecule complexes of the plurality of Cas9 molecule/gRNA molecule complexes; and   (c) determining one or more of the plurality of Cas9 molecule/gRNA molecule complexes is stable if the T m  value of the Cas9 molecule/gRNA molecule complex is greater than a T m  value of a reference molecule or a T m  reference value.   
     
     
         65 . The method of  claim 64 , wherein the T m  value is detected using a thermal shift assay. 
     
     
         66 . The method of  claim 65 , wherein the thermal shift assay is selected from differential scanning fluorimetry (DSF), differential scanning calorimetry (DSC), or isothermal titration calorimetry (ITC). 
     
     
         67 . The method of  claim 66 , wherein the Cas9 molecule/gRNA molecule complex is stable if the T m  value of the Cas9 molecule/gRNA molecule complex in the sample is at least 1° C., at least 2° C., at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C., at least 10° C., at least 11° C., at least 12° C., at least 13° C., at least 14° C., at least 15° C., at least 16° C., at least 17° C., at least 18° C., at least 19° C., or at least 20° C. greater than the T m  value of the reference molecule or the T m  reference value. 
     
     
         68 . The method of  claim 67 , wherein the reference molecule is selected from the group consisting of:
 (a) a reference Cas9 molecule in the absence of a gRNA molecule;   (b) a reference Cas9 molecule complexed with a gRNA molecule different from the first gRNA molecule in the complex being evaluated; and   (c) a reference Cas9 molecule/gRNA molecule complex formed under a different condition or buffer than the Cas9 molecule/gRNA molecule complex being evaluated.   
     
     
         69 . The method of  claim 68 , wherein the condition comprises using a proportion of Cas9 molecule and gRNA molecule relative to the complex being evaluated. 
     
     
         70 . A method of determining the stability of a Cas9 molecule/gRNA molecule complex, the method comprising:
 (a) combining a Cas9 molecule and a gRNA molecule in a sample to form the Cas9 molecule/gRNA molecule complex;   (b) detecting a melting temperature (T m ) value of the Cas9 molecule/gRNA molecule complex;   (c) measuring an activity value of the Cas9 molecule/gRNA molecule complex; and   (d) determining the Cas9 molecule/gRNA molecule complex is stable if (i) the T m  value of the Cas9 molecule/gRNA molecule complex is greater than a T m  value of a reference molecule or a T m  reference value and (ii) the activity value of the Cas9 molecule/gRNA molecule complex is greater than an activity value of a reference molecule or an activity reference value.   
     
     
         71 . The method of  claim 70 , wherein the activity comprises one or more of:
 an ability to induce indels;   an ability to modify a target DNA;   a propensity of a preselected repair method;   an ability of the gRNA molecule to remain hybridized to the DNA target; and   an ability of the gRNA molecule to bind to the Cas9 molecule of the Cas9 molecule/gRNA molecule complex.   
     
     
         72 . The method of  claim 71 , wherein the activity value is a binding value and the activity is the ability of the gRNA molecule to bind to the Cas9 molecule comprising:
 combining the gRNA molecule and the Cas9 molecule in a sample to form the Cas9 molecule/gRNA molecule complex;   measuring a binding value of the Cas9 molecule/gRNA molecule complex; and   determining the Cas9 molecule/gRNA molecule complex is stable if the binding value of the Cas9 molecule/gRNA molecule complex is greater than the binding value of a reference molecule or the binding reference value   
     
     
         73 . The method of  claim 72 , wherein the binding value is measured using a kinetics assay. 
     
     
         74 . The method of  claim 73 , wherein the kinetics assay is selected from surface plasmon resonance (SPR) assay, Bio-Layer Interferometry (BLI) assay, or gel band shift assay. 
     
     
         75 . The method of  claim 74 , wherein the T m  value is detected using a thermal shift assay. 
     
     
         76 . The method of  claim 75 , wherein the thermal shift assay is selected from differential scanning fluorimetry (DSF), differential scanning calorimetry (DSC), or isothermal titration calorimetry (ITC). 
     
     
         77 . The method of  claim 76 , wherein the Cas9 molecule/gRNA molecule complex is stable if the T m  value of the Cas9 molecule/gRNA molecule complex in the sample is at least 1° C., at least 2° C., at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C., at least 10° C., at least 11° C., at least 12° C., at least 13° C., at least 14° C., at least 15° C., at least 16° C., at least 17° C., at least 18° C., at least 19° C., or at least 20° C. greater than the T m  value of the reference molecule or the T m  reference value.