Journal: Genome Biology

Article Title: Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

doi: 10.1186/s13059-023-02928-7

Figure Lengend Snippet: Guide RNA exchange can be avoided by using different Cas species for KI and base editing. Graphical display of gRNA exchange between the Cas9 nuclease and the Cas9 base editor that can be prevented by applying a Cas12a nuclease in combination with a Cas9 base editor

Article Snippet: Cas9 Nuclease V3; IDT; 10 μg/μL = 61 μM) or Acidaminococcus sp. BV3L6 Cas12a (Alt-R A.s.Cas12a (Cpf1) Ultra; IDT; 10 μg/μL = 63 μM) are added and mixed by thorough pipetting.

Techniques:

Journal: Genome Biology

Article Title: Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

doi: 10.1186/s13059-023-02928-7

Figure Lengend Snippet: Eliminating Cas12a target sequence in the HDR template increases KI efficacy. a Design of the dsDNA template for TRAC targeted insertion is shown. The original template containing a mutated Cas9 PAM but an intact Cas12a PAM was modified by mutating the Cas12a PAM on the right homology arm to improve HDR efficiency by preventing cleavage of the repair template. b Representative flow cytometry histograms show KI efficiency using a template with an intact Cas12a PAM and either a Cas9 or Cas12a nuclease to target the TRAC gene. The CAR was stained by using an aFC antibody targeting the IgG1 hinge. c Knock-in efficiency quantified by flow cytometry using the intact or mutated template with a Cas9 nuclease (intact: n = 3, mutated: n = 5 individual donors; unpaired t -test). Empty shapes were performed with the old HDRT, filled shapes were performed with PAM-mutated HDRT. d Representative flow cytometry histograms show KI efficiency using a template with the mutated Cas12a PAM and either a Cas9 or Cas12a nuclease to target the TRAC gene. e Knock-in efficiency using the intact or mutated template with a Cas12a nuclease (intact: n = 3, mutated: n = 5 individual donors; unpaired t -test). f Fold change of KI efficiency with a donor template containing the mutated Cas12a PAM ( n = 5) in comparison to the mean ( n = 3) of the KI efficiency with the intact template (paired t -test). g Fold change increase of KI efficiency by using Cas12a instead of Cas9 as a nuclease with the HDRT with mutated PAM ( n = 5). Asterisks represent different p -values calculated in the respective statistical tests (ns: p ≥ 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001)

Article Snippet: Cas9 Nuclease V3; IDT; 10 μg/μL = 61 μM) or Acidaminococcus sp. BV3L6 Cas12a (Alt-R A.s.Cas12a (Cpf1) Ultra; IDT; 10 μg/μL = 63 μM) are added and mixed by thorough pipetting.

Techniques: Sequencing, Modification, Flow Cytometry, Staining, Knock-In, Comparison

Journal: Genome Biology

Article Title: Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

doi: 10.1186/s13059-023-02928-7

Figure Lengend Snippet: Co-delivery of Cas12a for KI and Cas9-derived BE avoids translocations during complex editing. a Experimental setup for the generation of CAR T cells by co-delivery of a Cas9 or Cas12a RNP mediating the TRAC insertion with sgRNAs directing an mRNA encoded ABE to target splice sites of the B2M and CIITA loci ( TRAC -CAR KI (Cas9) + MHC dKO (nCas9-BE), TRAC -CAR KI (Cas12a) + MHC dKO (nCas9-BE)). b Representative flow cytometry histograms show editing outcomes of TRAC -CAR KI (Cas9) + MHC dKO (nCas9-BE), TRAC -CAR KI (Cas12a) + MHC dKO (nCas9-BE) and mock electroporated cells. The CAR was stained by using an aFC antibody targeting the IgG1 hinge. c Summary plots for surface expression data from 5 donors. Empty shapes were performed with the old HDRT, filled shapes were performed with PAM-mutated HDRT. d Percentage of cells that are triple negative or positive for one, two, or all three analyzed surface markers as determined by applying flow cytometry based Boolean gating. e Frequencies of cells carrying balanced translocations as determined by ddPCR are shown for all six individual translocations and f as the sum of all translocations detected in mock, TRAC -CAR KI (Cas9) + MHC dKO (nCas9-BE) and TRAC -CAR KI (Cas12a) + MHC dKO (nCas9-BE) samples from n = 5 donors. Statistical analysis of flow cytometry and ddPCR data from 5 donors was performed using a one-way ANOVA of matched data with Geisser-Greenhouse correction. Multiple comparisons were performed by comparing the mean of each column with the mean of every other column and corrected by the Turkey test. Asterisks represent different p -values calculated in the respective statistical tests (ns: p ≥ 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001)

Article Snippet: Cas9 Nuclease V3; IDT; 10 μg/μL = 61 μM) or Acidaminococcus sp. BV3L6 Cas12a (Alt-R A.s.Cas12a (Cpf1) Ultra; IDT; 10 μg/μL = 63 μM) are added and mixed by thorough pipetting.

Techniques: Derivative Assay, Flow Cytometry, Staining, Expressing

Journal: Genome Biology

Article Title: Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

doi: 10.1186/s13059-023-02928-7

Figure Lengend Snippet: Amplicon sequencing confirms no indel formation at base-edited sites when combining Cas12a nuclease and Cas9 BE. Summary of CRIPResso2 analysis showing the frequency of total modified reads, frequency of indels, and quantification of intended base editing-mediated base changes mapped to B2M ( a ) or to CIITA ( b ). n = 5 healthy donors

Article Snippet: Cas9 Nuclease V3; IDT; 10 μg/μL = 61 μM) or Acidaminococcus sp. BV3L6 Cas12a (Alt-R A.s.Cas12a (Cpf1) Ultra; IDT; 10 μg/μL = 63 μM) are added and mixed by thorough pipetting.

Techniques: Amplification, Sequencing, Modification