Novel Type V-A CRISPR Effectors Are Active Nucleases with Expanded Targeting Capabilities

CRISPR J. 2020 Dec;3(6):454-461. doi: 10.1089/crispr.2020.0043. Epub 2020 Nov 3.

Abstract

Cas12a enzymes are quickly being adopted for use in a variety of genome-editing applications. These programmable nucleases are part of adaptive microbial immune systems, the natural diversity of which has been largely unexplored. Here, we identified novel families of Type V-A CRISPR nucleases through a large-scale analysis of metagenomes collected from a variety of complex environments, and developed representatives of these systems into gene-editing platforms. The nucleases display extensive protein variation and can be programmed by a single-guide RNA with specific motifs. The majority of these enzymes are part of systems recovered from uncultivated organisms, some of which also encode a divergent Type V effector. Biochemical analysis uncovered unexpected protospacer adjacent motif diversity, indicating that these systems will facilitate a variety of genome-engineering applications. The simplicity of guide sequences and activity in human cell lines suggest utility in gene and cell therapies.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacteria / genetics
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification*
  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / isolation & purification*
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / isolation & purification*
  • Endodeoxyribonucleases / metabolism*
  • Endonucleases / genetics
  • Gene Editing / methods*
  • Gene Editing / trends
  • Humans
  • Metagenomics / methods
  • Phylogeny
  • RNA, Guide, CRISPR-Cas Systems / genetics

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9
  • Cas12a protein
  • Endodeoxyribonucleases
  • Endonucleases