Engineered Cas12f Protein Expands Targetable Range via PAM Recognition
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Solution Overview
Problem
The Cas12f protein has limited targetable range due to its specific PAM sequence recognition, which restricts its genomic targeting capabilities, especially in complex eukaryotic genomes.
Innovation Solution
An engineered Cas12f protein with altered PAM recognition specificity is developed by introducing specific amino acid substitutions at defined positions, expanding its ability to recognize new PAM sequences and increase its genomic targeting range.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If the Cas12f protein uses its natural PAM recognition specificity (TTTR), then it maintains high cleavage efficiency at target sites, but its targetable range is limited to genomic sites with TTTR PAM sequences
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues (positions 170, 174, 184, 188, 191, 225, 230, and 272) in the Cas12f protein to alter its PAM recognition properties. These parameter changes in the protein sequence enable the Cas12f variant to recognize alternative PAM sequences (such as TTN, TTV, TTG, TAC, TAG, TAT, TGC, TGG, TGT, TCA, TCC, TCG, TCT) while maintaining functional activity, thus expanding the targetable genomic range without completely sacrificing recognition efficiency
Solution Approach 2:
The engineered Cas12f protein achieves universality by being capable of recognizing multiple different PAM sequences through the introduced amino acid substitutions. This multi-functional PAM recognition capability allows a single Cas12f variant to target diverse genomic locations that would otherwise require different Cas proteins or engineering approaches, making the system more versatile for gene editing applications across complex eukaryotic genomes
2Adaptability or versatility
If the Cas12f protein targets regions far from the PAM sequence, then it can access more genomic sites, but the indel efficiency decreases due to the editing window constraint
Solution Approach 1:
The patent applies parameter changes by mutating amino acid residues at positions 159 and 164 in the Cas12f protein to shift and expand the editing window. These parameter changes in the protein structure allow the catalytic domain to effectively reach and cleave DNA targets located further from the PAM sequence (up to 23 nucleotides away), thereby expanding the accessible editing window while maintaining sufficient indel efficiency for practical gene editing applications
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 engineered Cas12f protein variants exhibit enhanced indel efficiency and expanded targetable sites, enabling precise gene editing in regions previously inaccessible to the wild-type protein, thus broadening its therapeutic potential for genetic diseases.
Implementation Method 1
the Cas12f effector requires a specific PAM sequence for binding to target DNA... the wild-type Cas12f protein has preference for the PAM sequence TTTR... the engineered Cas12f protein exhibits altered PAM recognition specificity
Implementation Method 2
The CRISPR/Cas system... can edit or change a target sequence, for example, by site-specific DNA cleavage... the Cas12f effector... contains a RuvC nuclease domain... exhibit single-stranded DNA cleavage activity
Data Source
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AI summary
The present invention relates to an engineered Cas12 protein (for example, a Cas12f protein) having an expanded targeting range and use thereof. The engineered Cas protein comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of 159, 164, 170, 174, 184, 188, 191, 225, 230 and 272 of SEQ ID NO: 1 or at one or more amino acid positions corresponding thereto, and comprises an amino acid sequence having at least 80% identity to the sequence represented by SEQ ID NO: 1.