Cas12a Nuclease Variants for Expanded PAM Recognition
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR-Cas nucleases, such as Cas9, have stringent protospacer adjacent motif (PAM) specificities that limit the number of genomic target sites available for modification, restricting their usefulness in genome editing.
Innovation Solution
Development of modified Lachnospiraceae bacterium CRISPR-Cas12a (LbCas12a) nucleases with altered PAM recognition specificity by introducing mutations at specific positions, allowing for broader recognition of PAM sequences, and the use of a CRISPR-Cas system comprising a modified LbCas12a polypeptide and a guide nucleic acid to target and modify nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Cas9 nuclease is used with its natural PAM specificity (NGG motif), then the nuclease can efficiently recognize and cut target sites with this specific PAM sequence, but the number of available genomic target sites is limited due to stringent PAM requirements
Solution Approach 1:
The patent applies parameter changes by introducing amino acid mutations at specific positions (K116, K120, K121, D122, E125, T148, T149, T152, D156, E159, Q529, G532, D535, K538, D541, Y542, L585, K591, M592, K595, V596, S599, K600, K601, Y616, Y646, and/or W649) in the LbCas12a nuclease protein. These mutations alter the PAM recognition parameters of the nuclease, enabling it to recognize a broader range of PAM sequences beyond the natural TTTV specificity, thereby increasing the number of available genomic target sites while maintaining reliable PAM recognition.
2Reliability
If LbCas12a nuclease with natural TTTV PAM specificity is used, then the nuclease maintains high specificity for its natural PAM sequence, but the versatility for targeting different genomic locations is reduced
Solution Approach 1:
The patent implements universality by creating a multi-functional PAM recognition capability in the LbCas12a nuclease. Through the introduced amino acid mutations, the nuclease can recognize multiple different PAM sequences (including but not limited to TTTV, TTTC, TTTA, TTTG, and other variations), making it universally applicable to a wide range of genomic target sites while maintaining reliable recognition through the guide nucleic acid pairing mechanism.
3Adaptability or versatility
If mutations are introduced at multiple positions in the LbCas12a protein to alter PAM specificity, then the nuclease can recognize broader PAM sequences, but the complexity of characterizing and validating the mutant variants increases
Solution Approach 1:
The patent applies segmentation by dividing the PAM recognition interface into specific functional regions corresponding to the mutated amino acid positions. This allows the complex task of characterizing multiple variants to be segmented into manageable analyses of individual mutation sites (K116, K120, K121, etc.) and their individual contributions to PAM recognition, facilitating systematic validation and selection of optimal variants.
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 modified LbCas12a nucleases exhibit improved PAM specificity, expanding the range of genomic target sites accessible for modification, enhancing the versatility and efficiency of genome editing processes.
Implementation Method 1
the spacer sequence is capable of hybridizing to a target nucleic acid, thereby guiding the modified LbCas12a polypeptide and the polypeptide of interest to the target nucleic acid
Data Source
AI summary
This invention relates to variants of Cas12a nucleases having altered protospacer adjacent motif recognition specificity. The invention further relates to methods of making CRISPR-CAS nuclease variants and methods of modifying nucleic acids using the variants.