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1.
Nat Commun ; 13(1): 2833, 2022 05 20.
Article in English | MEDLINE | ID: mdl-35595757

ABSTRACT

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.


Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Cas Systems/genetics , Endonucleases/genetics , Endonucleases/metabolism , Gene Editing/methods , HEK293 Cells , Humans , RNA/metabolism , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism
2.
Methods Enzymol ; 659: 371-389, 2021.
Article in English | MEDLINE | ID: mdl-34752296

ABSTRACT

Cell-free protein synthesis is a powerful tool to produce recombinant proteins, and as an open system, can often integrate all or part of downstream assays. Here we describe in vitro synthesis of the Streptococcus pyogenes type II-A CRISPR-Cas9 ribonucleoproteins (SpCas9 RNPs), consisting of the effector protein and the single guide RNAs (sgRNAs). In spite of its large molecular weight (160kDa), the SpCas9 effector is expressed relatively well from linear DNA templates under T7 promoter in commercial reconstituted cell-free protein synthesis systems. sgRNAs can be added before the effector synthesis reaction, or transcribed directly from linear DNA templates during the synthesis reaction. The newly synthesized SpCas9 effector forms an active RNP complex with sgRNAs. When a reconstituted cell-free system is used, the target DNA templates can also be added in the reactions, thereby combining CRISPR-Cas synthesis and activity assay.


Subject(s)
Gene Editing , Ribonucleoproteins , CRISPR-Cas Systems , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Streptococcus pyogenes/genetics
3.
ACS Nano ; 15(1): 489-502, 2021 01 26.
Article in English | MEDLINE | ID: mdl-33370106

ABSTRACT

DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for specific properties gives the opportunity to identify polymerases with different features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to an α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of ∼100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with altered physical properties.


Subject(s)
Nanopores , DNA , DNA-Directed DNA Polymerase , Kinetics , Sequence Analysis, DNA
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