Double-Tap Genome Editing Using Secondary gRNAs
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Solution Overview
Problem
Current genome editing methods, particularly CRISPR-based systems, face challenges in precision and efficiency, especially in mammalian cells and mouse germline transformations, where HDR frequencies are low and resistance alleles hinder gene drive spread.
Innovation Solution
The 'double-tap' method employs additional gRNAs to target high-frequency indel products created by end joining pathways during HDR events, providing a second opportunity for HDR processing and recycling resistance alleles to enhance gene drive efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-based genome editing is performed using standard HDR methods, then precision editing can be achieved, but editing efficiency is low due to low HDR frequencies in mammalian cells
Solution Approach 1:
The method performs preliminary characterization of indel products before the main editing process. By identifying and characterizing the sequences of common indel products in advance, the system prepares secondary gRNAs that can target these specific indel sequences, creating a staged approach that increases the likelihood of successful HDR editing on subsequent attempts
Solution Approach 2:
The double-tap method extends the editing process by providing continuous opportunities for HDR. Instead of a single HDR attempt, the system implements multiple sequential opportunities: the initial DSB creates indel products, and secondary gRNAs continuously target these indel products for additional HDR attempts, maintaining the useful action of HDR processing throughout the extended time window
2Productivity
If additional gRNAs are used to target indel products, then HDR efficiency is improved, but system complexity increases
Solution Approach 1:
The method creates simplified copies or representations of the target sequences. By characterizing indel products and designing secondary gRNAs that copy the targeting strategy of primary gRNAs but against indel-specific sequences, the system manages complexity through systematic replication of the gRNA design principle rather than arbitrary addition of components
Solution Approach 2:
The system changes the target parameter of gRNAs from wild-type sequences to indel-containing sequences. This parameter change allows the same Cas9-gRNA mechanism to be reused with modified specificity, improving efficiency without fundamentally changing the system architecture or requiring entirely new molecular components
3Productivity
If resistance alleles are generated during gene drive, then gene drive spread is blocked, but the double-tap method recycles these alleles to improve spread
Solution Approach 1:
The method converts the harmful effect of resistance allele formation into a beneficial opportunity for further gene drive spread. By designing secondary gRNAs that specifically target resistance alleles, the system transforms these previously blocking mutations into new targets that can be converted back to functional alleles through HDR, thereby converting the harm of resistance into the benefit of additional conversion opportunities
Solution Approach 2:
The double-tap method implements recovery of discarded genetic material. Resistance alleles are typically considered discarded or failed outcomes that block gene drive propagation. The system recovers these discarded alleles by using secondary gRNAs to target them for HDR-mediated conversion back to functional drive alleles, thereby recovering potentially useful genetic material that would otherwise be lost
Data Source
AI summary
The present disclosure provides a “double tap” method to improve genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The “double tap” method uses multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary or tertiary or subsequent gRNAs that target the most common indel sequence(s), which provides a “second/third chance” at editing. The “double tap” method also improves gene drive efficiency by recycling resistance alleles. The “double tap” method can be readily implemented in any CRISPR-based gene drive and in a subject that has HDR as a DNA repair mechanism and/or a system suffering from low HDR frequencies to improve performance by boosting efficient gene editing.