Novel AsCpf1 Mutations for Higher DNA Cleavage at TTTT PAM Sites
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Solution Overview
Problem
The Cas12a system has limited utility due to its relatively low enzymatic activity and restricted PAM compatibility, making it less efficient for genome editing compared to Cas9, particularly at AT-rich sites, and requires improvement for broader application in eukaryotic cells.
Innovation Solution
Development of novel AsCpf1 and AsCas12a variants, such as M537R, F870L, and M537R/F870L, which enhance DNA cleavage activity at both canonical and non-canonical PAM sites, and include nuclear localization signals for efficient delivery to eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If Cas12a is used for genome editing, then AT-rich sites can be targeted that are inaccessible to Cas9, but the enzymatic activity is relatively low resulting in lower editing efficiency
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (M537R, F870L) in the Cas12a protein sequence to alter its enzymatic properties. These parameter changes in the protein structure enable enhanced DNA cleavage activity while maintaining PAM recognition flexibility, thereby resolving the contradiction between versatility and productivity
Solution Approach 2:
The patent creates composite functional elements by combining multiple mutations (M537R/F870L double mutant) and fusing Cas12a with nuclear localization signals (NLS) to create a enhanced enzyme system. This composite approach allows the modified Cas12a to achieve both high enzymatic activity and cellular delivery capability, overcoming the limitations of the wild-type enzyme
2Adaptability or versatility
If Cas12a is used instead of Cas9, then broader PAM site compatibility is achieved, but the overall genome editing efficiency is reduced due to low enzymatic activity
Solution Approach 1:
Specific amino acid substitutions (M537R, F870L) are introduced to change the enzymatic parameters of Cas12a, enhancing its DNA cleavage activity while preserving its ability to recognize diverse PAM sequences. This allows the enzyme to maintain its advantage in targetable loci expansion while achieving reliable editing efficiency
Solution Approach 2:
The patent introduces nuclear localization signals (NLS) as intermediary elements that facilitate the delivery of Cas12a into the eukaryotic nucleus. This intermediary component enables the enzyme to function effectively in eukaryotic cells, bridging the gap between bacterial origin and eukaryotic application, thereby improving overall genome editing reliability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The enhanced variants significantly improve DNA cleavage efficiency at TTTT PAM sites, broadening PAM flexibility and enabling efficient genome editing in human cells, surpassing the capabilities of wild-type Cas12a.
Implementation Method 1
Cas12a is an RNA-guided endonuclease found in bacterial species including Acidaminococcus sp. and is part of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) adaptive immune system. The Cas12a-gRNA ribonucleoprotein (RNP) complex mediates double-stranded DNA breaks (DSBs)
Implementation Method 2
include nuclear localization signals for efficient delivery to eukaryotic cells
Data Source
AI summary
The present disclosure concerns polynucleotides and amino acids of Acidaminococcus sp. Cas12a (Cpf1) and methods for their use for genome editing in eukaryotic cells.