Engineered cas effector proteins and methods of use thereof
An effector protein, engineering technology, applied in the field of a method and composition for engineering Cas effector protein
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Embodiment 1
[0487] Example 1: Engineering pipelines of enzymes with improved efficiency
[0488] This example provides a strategy to design Cas enzymes with enhanced kinetics of conformational transitions that result in better catalytic efficiency of Cas endonucleases. Exemplary methods provided herein allow for the engineering of Cas proteins with improved activities (eg, target binding, double-strand cleavage activity, nickase activity, and / or gene editing activity).
[0489] We believe that the conditions for Cas conformational transitions in human cells may not be optimal, and that enhanced conformational transition kinetics may lead to better catalytic efficiency of Cas enzymes. To design Cas proteins with enhanced kinetics of conformational transitions, we devised a strategy to increase the flexibility of the flexible region in Cas enzymes. Figure 1 shows the pipeline used to design SaCas9 variants as an example of this design workflow.
[0490] Enhanced Cas enzyme design pipeli...
Embodiment 2
[0512] Example 2: Design and characterization of engineered BhCas12b
[0513] This example describes the use of the design pipeline described in Example 1 to design and characterize Bacillus somura with improved gene editing activity in human cells ( Bacillus hisashii ) An engineered variant of Cas12bv4 (BhCas12bv4).
[0514] The flexible region of Cas12bv4 was determined in silico using DynaMine as described in Example 1 above. Peaks aa and candidate flexible regions for engineering were identified as described in Example 1 above. figure 2 The flexibility (S2 score) spectrum of BhCas12bv4 is shown, with selected peaks aa indicated by circles. Table 1 below shows the flexible region sequence (SEQ ID NO: 81) and Y>G substitutions of the engineered variants (SEQ ID NO: 82). Amino acid positions are based on SEQ ID NO: 1. BhCas12bv4 has no resolved crystal structure, but the highly homologous BthCas12b (>98% homology) has a crystal structure available, so the linker of BhCa...
Embodiment 3
[0518] Example 3: Design and characterization of engineered Cas12i2
[0519] This example describes the use of the design pipeline described in Example 1 to design and characterize Cas12i2 engineered variants with improved gene editing activity in human cells.
[0520] There is no known 3D structure of Cas12i2, and no equivalent of its structure has been resolved. Therefore, we sought to test whether our method, which does not require structure solving, can be used to engineer Cas12i2 variants with improved activities such as target binding, double-strand cleavage activity, nickase activity and / or gene editing activity.
[0521] The flexible region of Cas12i2 was determined in silico using DynaMine as described in Example 1 above. The flexibility (S2 score) spectrum of Cas12i2 is as Figure 4 shown. Based on the S2 score spectrum, we selected peaks with S2 scores less than 0.71 as flexible regions. Although there is no resolved structural information for Cas12i2 or closely...
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