Journal: iScience

Article Title: High-level correction of the sickle mutation is amplified in vivo during erythroid differentiation

doi: 10.1016/j.isci.2022.104374

Figure Lengend Snippet: Correction of the sickle mutation in long-term xenografted hematopoietic cells (A) Schematic depicting the β-globin gene ( HBB ) with the targeted region enlarged. Sequence shown in black is the sickle allele. The G10 guide RNA (red line) targets Cas9 cleavage to a site near the sickle mutation. The 168-base single-stranded DNA oligonucleotide donor induces sequence changes shown in red. HDR tract conversion proceeds from the Cas9 cleavage site (red arrow), alters the PAM motif to prevent cleavage of the edited allele, but does not always extend to the site of the sickle mutation. (B) Schematic outlining the large-scale xenografting experiment, and analysis of the engrafted cells.

Article Snippet: Wild-type Cas9 protein, Cas9 HF-1, and Cas9 espCas9-1.1 were obtained from the Berkeley Macro Lab. AltR HiFi Cas9 was purchased from IDT, Inc. or Aldevron, Inc.

Techniques: Mutagenesis, Sequencing

Journal: iScience

Article Title: High-level correction of the sickle mutation is amplified in vivo during erythroid differentiation

doi: 10.1016/j.isci.2022.104374

Figure Lengend Snippet: Assessment of off-target cleavage by the Cas9 RNP (A) Representative GUIDE-seq with the Cas9 RNP in K562 cells (left), and genomic coordinates of GUIDE-seq hits detected in three independent replicates (table at right). (B) Comparison of editing at HBB by high-fidelity Cas9 variants espCas9-1.1 and Alt-R HiFi Cas9 in healthydonor HSPCs. Error bars depict standard deviation from the mean. (C) GUIDE-seq in CD34 + HSPCs edited with the 3xMS-G10 RNP with wild-type Cas9. Only two sites, the on-target site in HBB and the primary off-target site OT1, are detected. (D) Representative GUIDE-seq with Alt-R HiFi Cas9 RNP in K562 cells. (E) Total gene editing rates (%HDR + %NHEJ) measured by pooled-primer PCR at 190 of 201 identified off-targets, in the edited HSPCs injected into Cohorts one and 2 (“input” in C), and in healthy donor HSPCs edited with Alt-R HiFi Cas9; indels observed at the same sites in untreated cells are subtracted. Blue dots: on-target HBB ; green dots: OT1; orange dots: OT-II. Editing at >0.2% of alleles (dashed line) by wild-type Cas9 is observed only at HBB , OT1, and OT-II, and by high-fidelity Cas9 only at HBB and OT1.

Article Snippet: Wild-type Cas9 protein, Cas9 HF-1, and Cas9 espCas9-1.1 were obtained from the Berkeley Macro Lab. AltR HiFi Cas9 was purchased from IDT, Inc. or Aldevron, Inc.

Techniques: Standard Deviation, Injection

Journal: iScience

Article Title: High-level correction of the sickle mutation is amplified in vivo during erythroid differentiation

doi: 10.1016/j.isci.2022.104374

Figure Lengend Snippet:

Article Snippet: Wild-type Cas9 protein, Cas9 HF-1, and Cas9 espCas9-1.1 were obtained from the Berkeley Macro Lab. AltR HiFi Cas9 was purchased from IDT, Inc. or Aldevron, Inc.

Techniques: Isolation, Recombinant, Mutagenesis, Sequencing, Software

Journal: Microbial Biotechnology

Article Title: Developing a CRISPR‐assisted base‐editing system for genome engineering of Pseudomonas chlororaphis

doi: 10.1111/1751-7915.14075

Figure Lengend Snippet: Design of a CRISPR‐assisted base‐editing system tailored for Pseudomonas chlororaphis . A. Schematic illustration of the plasmid‐borne base‐editing system. The tool consists of an enhanced specificity Cas9 nickase (eSpCas9pp D10A ), fused to a cytidine deaminase (rAPOBEC1, which catalyses the C‐to‐T conversion), and the uracil DNA glycosylase inhibitor (UGI). The base‐editing machinery is directed to the target DNA by a single guide RNA (sgRNA). PAM , protospacer adjacent motif. B. Biochemical reaction catalysed by the cytidine deaminase rAPOBEC1. C. Structure of plasmid pBRC1, containing the construct that encodes the SpCas9pp D10A , rAPOBEC1 and UGI fusion. This module is under transcriptional control by the L‐arabinose‐inducible AraC/ P araBAD system, whereas the sgRNA is constitutively expressed from a P lac promoter. XTEN, short peptide linker sequence with no specific structure; N 20 , specific 20‐nt sequence targeting the gene to be edited; and Km R , kanamycin resistance. D. Strategy followed for gene inactivation by introducing premature STOP codons (TAA, TAG and TGA) in target genes using the base‐editing system. UGI inhibits uracil base excision on the non‐template DNA strand, so that DNA repair occurs on the template strand.

Article Snippet: A DNA cassette encoding the cytidine deaminase from rat [rAPOBEC1; Komor et al . ( )], an enhanced specificity Cas9 nickase [eSpCas9pp D10A , carrying the point mutations K848A, K1003A, R1060A and D10A; Sun et al . ( )], a XTEN linker [a short connector sequence; Komor et al . ( )] and the uracil DNA glycosylase inhibitor (UGI) was amplified from plasmid pSEVA2BE [a derivative of standard vector pSEVA644; Sun et al . ( )] and assembled in plasmid pBRC1 (deposited in Addgene, ID 183064).

Techniques: CRISPR, Plasmid Preparation, Construct, Control, Sequencing