Type V Cas Proteins for AAV-Compatible Genome Editing
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Solution Overview
Problem
Existing CRISPR-Cas9 systems face challenges such as limited packaging capacity in AAV vectors, target sequence specificity, and varying DNA cut types, which affect genome editing efficiency and precision.
Innovation Solution
Development of Type V Cas proteins from diverse unclassified bacteria, offering amino acid sequences with high identity to specific SEQ IDs, and potential fusion proteins with additional domains for enhanced functionality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Cas9 is used for genome editing, then editing capability is achieved, but packaging capacity of AAV vector is exceeded due to large size
Solution Approach 1:
The patent applies parameter changes by switching from Class 2 Type II Cas9 to Class 2 Type V Cas12a, which has a smaller molecular weight (1028 amino acids vs. larger Cas9). This parameter change in protein size enables packaging within AAV vectors while maintaining genome editing capability. The Type V Cas system provides an alternative parameter set that resolves the contradiction between editing functionality and deliverable size.
2Ease of manufacture
If Cas9 creates blunt ends, then simple cutting is achieved, but flexibility for gene insertions and substitutions is limited
Solution Approach 1:
The patent changes the cutting pattern parameter from blunt ends (Cas9) to staggered ends (Type V Cas12a). This parameter change in DNA end configuration provides enhanced versatility for gene insertions and substitutions while maintaining the fundamental genome editing function. The staggered cut pattern creates overhangs that facilitate various genetic manipulation strategies.
3Manufacturing precision
If Cas9 produces small indels, then precise point mutations are achieved, but removal of target sequences is limited
Solution Approach 1:
The patent changes the deletion size parameter from small indels (Cas9) to mid-sized deletions (Type V Cas12a, generally tens of nucleotides). This parameter change enables effective removal of target sequences such as transcription factor binding sites and splice sites while maintaining precision at the target locus. The increased deletion capacity provides versatility for both point mutations and larger sequence removals.
4Adaptability or versatility
If Type V Cas proteins target T-rich sequences, then access to different genomic regions is achieved, but target sequence specificity is reduced
Solution Approach 1:
The patent changes the PAM sequence parameter to T-rich sequences for Type V Cas12a, which expands genomic region accessibility compared to Cas9 targets. This parameter change allows targeting of diverse genomic locations while the crRNA guide sequence maintains specificity through complementary base pairing. The system balances broad accessibility with precise targeting through the combination of relaxed PAM requirements and sequence-specific crRNA guidance.
Data Source
AI summary
Type V Cas proteins, for example Type V Cas proteins referred to as ZWGD, ZJHK, ZIKV, ZZFT, YYAN, ZZGY, ZKBG, ZZKD, ZXPB, ZPPX, ZXHQ, ZQKH, ZRGM, ZTAE, ZSQQ, ZSYN, ZRBH, ZWPU, ZZQE, and ZRXE Type V Cas proteins; gRNAs for Type V Cas proteins; systems comprising Type V Cas proteins and gRNAs; nucleic acids encoding the Type V Cas proteins, gRNAs and systems; particles comprising the foregoing; pharmaceutical compositions of the foregoing; and uses of the foregoing, for example to alter the genomic DNA of a cell.