Journal: bioRxiv

Article Title: Efficient repair of human homozygous genetic mutation by CRISPR/Cas9 mediated interlocus gene conversion

doi: 10.1101/2022.09.05.506576

Figure Lengend Snippet: a , Seven sgRNAs targeting the β 0 4142 core marked with green arrows. CD4142 deletion base pairs marked with chromatic rectangles. b , Schematic diagram of CRISPR/Cas9 targeting sites in the mutant HBB gene. Blue lines label sgRNA sequences; purple lines label exogenous DNA templates. c , Editing efficiency of Cas9 RNPs coupled with the various sgRNAs in CD34 + HSPCs from homozygotes measured by TIDE analysis. HM mock represents only CD34 + HSPCs from homozygous Donor #1; Cas9 only represents CD34 + HSPCs from homozygous Donor #1 electroporated with Cas9 only. Error bars indicate the standard deviation ( n = 3 replicates); donors edited without sgRNA were plotted as a negative control. d , CD34 + cells from patients #1 and #2 were electroporated with Cas9 coupled with sgRNA-1 and with different concentration gradient DNA templates. HM mock, HSPCs of homozygous Donor #1 only; HT mock, HSPCs of heterozygote Donor #2 only; w/o donor, patient donors edited with RNPs but without DNA templates; Forward and reverse normal gene sequences were synthesized expressed by w/ssODN and w/ssODN-R. These two single-stranded DNAs were incorporated into dsODNs annealed by PCR. Primers with 5′ modifications included an amine group with a C6 linker (AmC6) or C12 linker (AmC12) expressed by w/ss-AmC6, w/ss-AmC12. e , β globin expression by RT–qPCR analysis in erythroid cells in vitro differentiated from RNP-edited CD34 + HSPCs. Data are plotted as the mean ± s.d. and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.

Article Snippet: We transformed pET-21a _ 3×NLS–Streptococcus pyogenes Cas9 available on Addgene (SpCas9, ID #114365) into BL21 (DE3) chemically competent cells (TransGen Biotech, CD601-02) and grew the cells in LB medium at 37°C 220 rpm until the density reached OD600 = 2-2.5.

Techniques: CRISPR, Mutagenesis, Standard Deviation, Negative Control, Concentration Assay, Synthesized, Expressing, Quantitative RT-PCR, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Efficient repair of human homozygous genetic mutation by CRISPR/Cas9 mediated interlocus gene conversion

doi: 10.1101/2022.09.05.506576

Figure Lengend Snippet: a , Efficiency of restoration edited by Cas9 coupled with the various sgRNAs in homozygous β 0 4142 CD34 + HSPCs measured by deep-seq analysis. Error bars indicate the standard deviation ( n =3 replicates). b , β-globin expression by RT–qPCR analysis in erythroid cells in vitro differentiated from RNP-edited CD34 + HSPCs. c , The homologous genomic sequence of HBB and HBD , Red triangle represents the deletion base pairs, the color purple marked a NGG protospacer-adjacent motif, gray marked sgRNA-1. d , Restoration of the β 0 4142 in HBD-4142 KO donor determined by deep-seq analysis versus edited only by the RNP complex. Gray points HBD - represent samples with KO of HBD-4142 edited by Cas9 coupled with sgRNA-1 72 hours later, and red points represent samples edited only with Cas9 coupled with sgRNA-1. e , Efficiency of restorations as measured by deep-seq analysis of Cas9:sgRNA-1 RNP targeting the β 0 4142 site in CD34 + HSPCs from patient donors (see details in Table S2). f–h , Genotyping of erythroid cells derived from single colonies screened out edited CD34 + HSPCs from Donors #1 and #2. The purple dashed line in f highlights IVS1-1 G>T, corrected deletions of β 0 4142 (orange dashed line), specific fragments or base pairs of HBB distinct from HBD (blue dashed line in g, h ). i , Indels at the HBD locus measured by deep-seq analysis of sgRNA-1 RNP targeting the β 0 4142 site. In all graphs, data are plotted as the mean ± s.d. and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.

Article Snippet: We transformed pET-21a _ 3×NLS–Streptococcus pyogenes Cas9 available on Addgene (SpCas9, ID #114365) into BL21 (DE3) chemically competent cells (TransGen Biotech, CD601-02) and grew the cells in LB medium at 37°C 220 rpm until the density reached OD600 = 2-2.5.

Techniques: Standard Deviation, Expressing, Quantitative RT-PCR, In Vitro, Sequencing, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Efficient repair of human homozygous genetic mutation by CRISPR/Cas9 mediated interlocus gene conversion

doi: 10.1101/2022.09.05.506576

Figure Lengend Snippet: CD34 + HSPCs from donors with β 0 4142 were electroporated with 3NLS Cas9 and sgRNA-1 dependent or independent of exogenous DNA templates. We transplanted 5–8 × 10 5 treated cells into NBSGW mice via tail-vein injection. Mouse BM was collected and analyzed 16 weeks after transplantation. a , Experimental workflow. b , In BM, as well as the indel frequencies determined by TIDE analysis. c , Engraftment measured by the percentage of human CD45 + (hCD45 + /(hCD45 + +mCD45 + )) cells in recipient mouse BM. d , Human B cells (hCD19 + ) and myeloid cells (hCD33 + ) as percentages of the hCD45 + population in recipient BM. e , Human erythroid precursors (hCD235a + ) as a percentage of human and mouse CD45 − cells in recipient BM. f , In BM, proportions of β-globin mRNA expression level by RT–qPCR normalized by α-globin. g , β 0 4142-to-normol editing efficiency in human CD34 + cell-derived lineages from recipient BM. h . Indel spectrum of input cells from Donor #1 and Donor #5 electroporated with sgRNA-1 before transplantation and BM-engrafted human cells 16 weeks after transplantation. The indel spectrum was determined by deep sequencing analysis. These data comprise 3 mice transplanted from Donor #5 and 5 mice transplanted from Donor #1 with sgRNA-1 edited inputs. Each symbol represents a mouse, and the mean for each group is shown. The median of each group with 3–5 mice in b, c, e, and f is shown as a line. Data are plotted as the mean ± s.d. for d, g and were analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.

Article Snippet: We transformed pET-21a _ 3×NLS–Streptococcus pyogenes Cas9 available on Addgene (SpCas9, ID #114365) into BL21 (DE3) chemically competent cells (TransGen Biotech, CD601-02) and grew the cells in LB medium at 37°C 220 rpm until the density reached OD600 = 2-2.5.

Techniques: Injection, Transplantation Assay, Expressing, Quantitative RT-PCR, Derivative Assay, Sequencing, Two Tailed Test

Journal: Nature communications

Article Title: MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits.

doi: 10.1038/s41467-020-19842-2

Figure Lengend Snippet: Fig. 3 MiCas9 reduces off-target indel rates. a Off-target and on-target indel rates associated with sg1-VEGFA by spCas9 and miCas9 pDNAs. b Off- target and on-target indel rates associated with sg-FANCF2 by spCas9 and miCas9 pDNAs. c Indel rates at Guide-seq predicted potential off-target loci associated with sg1-VEGFA by pDNAs of different nucleases. d Indel rates at Guide-seq predicted potential off-target loci associated with sg-FANCF2 by pDNAs of different nucleases. Sp: spCas9, Mi: miCas9, Hy: hypaCas9, Hi: HiFiCas9, HE: Cas9-HE, GE: Cas9-GE, NC: negative control with non-specific gRNA. #Reads: Average amplicon reads per sample. BD: below detection, representing values <0.10%. Three independent experiments were performed for each condition. Data are presented as mean ± standard error of means (SEM) in a and b, and as heat map in c and d. Unpaired t-test (two tailed) was used to compare data using GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA). Source data are available in the Source Data file.

Article Snippet: HypaCas9 (Cat# 101178), Cas9HE (Cat# 109400), and Cas9-GE (Cat# 109401) plasmid DNAs were acquired from Addgene (www.addgene.org).

Techniques: Negative Control, Amplification, Two Tailed Test, Software