Engineered Guide RNA for CRISPR-Cpf1 Editing
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Solution Overview
Problem
The CRISPR-Cpf1 genome editing system has low gene editing frequencies, necessitating the development of new methods to modify guide RNA sequences for improved efficiency.
Innovation Solution
The use of engineered guide RNAs with extensions at the 5′ and/or 3′ ends and chemical modifications to the nucleotides, including changes to the nucleobase, ribose sugar, and phosphodiester linkage, to enhance the CRISPR-Cpf1 genome editing system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard guide RNA sequences are used in the CRISPR-Cpf1 system, then the system structure is simple, but the gene editing efficiency is low
Solution Approach 1:
The guide RNA is divided into distinct functional segments: a 5' extension region (nucleotides 1-50) that enhances stability and binding, a stem loop structure (nucleotides 51-100) that facilitates proper folding and Cpf1 interaction, and a 3' guide segment (nucleotides 101-150) that hybridizes with the target DNA sequence. This segmentation allows each region to be optimized independently for its specific function, thereby improving overall gene editing efficiency while maintaining a manageable structural complexity
Solution Approach 2:
The guide RNA employs composite chemical modifications including 2'-O-methyl modifications at specific positions, phosphorothioate linkages at the 5' end, and unlocked nucleic acid (UNA) residues at periodic intervals. This composite approach combines different chemical properties (enhanced stability from phosphorothioates, improved binding from 2'-O-methyl, and enhanced flexibility from UNA) to create a guide RNA that achieves superior gene editing efficiency compared to unmodified RNA
2Productivity
If guide RNA sequences are modified to improve editing efficiency, then the gene editing frequency increases, but the manufacturing complexity increases
Solution Approach 1:
The guide RNA synthesis employs systematic parameter changes including: (1) incorporating 2'-O-methyl modifications at positions 5-10 and 145-150 to enhance stability without requiring complete sequence redesign, (2) adding phosphorothioate linkages at the 5' terminal positions (1-5) to improve nuclease resistance, and (3) inserting UNA residues at positions 20, 40, 60, 80, and 100 to enhance binding flexibility. These targeted parameter changes achieve improved gene editing frequency while maintaining compatibility with standard oligonucleotide synthesis protocols
Solution Approach 2:
The guide RNA is designed with pre-calculated optimal lengths and modification patterns that can be directly ordered from commercial synthesizers. The 5' extension region (nucleotides 1-50) and stem loop structure (nucleotides 51-100) are designed in advance with specific secondary structures that facilitate proper folding, eliminating the need for post-synthesis folding optimization. This preliminary design approach simplifies manufacturing by allowing direct synthesis of the final functional product
3Reliability
If chemical modifications are added to the guide RNA, then the stability and efficiency improve, but the cost and complexity of synthesis increase
Solution Approach 1:
Chemical modifications are applied locally at specific positions rather than uniformly throughout the guide RNA sequence. 2'-O-methyl modifications are placed at positions 5-10 and 145-150 where they provide maximum stability enhancement. Phosphorothioate linkages are concentrated at the 5' terminal positions (1-5) where they protect against exonuclease degradation. UNA residues are positioned at intervals (20, 40, 60, 80, 100) to provide localized flexibility without requiring modifications at every position. This local quality approach achieves improved reliability with minimal synthesis complexity
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 guide RNAs significantly improve the gene editing efficiency of the CRISPR-Cpf1 system, as demonstrated by increased cleavage efficiency in T7E1 assays.
Implementation Method 1
a guide RNA comprising a guide segment that hybridizes with a target sequence of a DNA molecule in a eukaryotic cell to form an RNA/DNA hybrid
Implementation Method 2
the Cpf1 protein cleaves the DNA molecule
Data Source
AI summary
The present disclosure generally relates to systems, methods and compositions for use in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cpf1 genome editing systems.