Inference of CRISPR Edits from Sanger Trace Data

Hsiau, Tim; Conant, David S.; Rossi, Nicholas A. A.; Maures, Travis J.; Waite, Kelsey; Yang, Jason; Joshi, Sahil; Kelso, Reed; Holden, Kevin; Bl, Enzmann; Stoner, Rich

doi:10.1101/251082

Cited by 322 publications

(351 citation statements)

References 5 publications

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“…The sgRNA designed and tested to target IL-6R and Spy Cas9 complexed into a ribonucleoprotein (RNP) and the ssODN designed to correct IL-6R was electroporated into the patient T cells. Genomic (g)DNA was extracted from half of the cells and Sanger Sequenced (11).…”

Section: Resultsmentioning

confidence: 99%

The use of CRISPR for variant specificity in the genetic diagnosis of primary immunodeficiency disease (PID)

Mann

Smith

Spencer

et al. 2019

Preprint

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The functional validation of genetic variants of uncertain significance (VUS) found in PID patients by next-generation sequencing has traditionally been carried out in model systems that are susceptible to artefact. We use CRISPR correction of primary human T lymphocytes to demonstrate that a specific variant in an IL-6R deficient patient is causative for their condition.This methodology can be adapted and used for variant assessment of the heterogeneous genetic defects that affect T lymphocytes in PID.

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Section: Resultsmentioning

confidence: 99%

The use of CRISPR for variant specificity in the genetic diagnosis of primary immunodeficiency disease (PID)

Mann

Smith

Spencer

et al. 2019

Preprint

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“…An alternative to testing sgRNA and/or the knock‐in mutation efficiency with AS‐PCR would be to sequence amplicons from mutated bulk cells and analyse by software such as ICE or TIDE …”

Section: Discussionmentioning

confidence: 99%

“…An alternative to testing sgRNA and/or the knock-in mutation efficiency with AS-PCR would be to sequence amplicons from mutated bulk cells and analyse by software such as ICE 39 or TIDE. 40 However, in our opinion, developing an AS-PCR set-up for screening of a large number of clones with possible knock-in mutations is generally easy to do and more affordable than Sanger sequencing.…”

Section: Discussionmentioning

confidence: 99%

A simple and efficient workflow for generation of knock‐in mutations in Jurkat T cells using CRISPR/Cas9

et al. 2020

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CRISPR/Cas9 is a powerful gene‐editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9‐mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock‐in mutations were introduced in Jurkat T cells by homologous directed repair using single‐stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus‐dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele‐specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock‐in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.

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“…To gain a more quantitative understanding of the cleavage activity of Mad7 at the chosen target loci, PCR amplicons of cells transfected with Mad7 targeting the respective loci (AAVS1, CCR5, TRAC) were submitted for Sanger sequencing and subjected to the bioinformatic ICE analysis program that infers CRISPR activity from sequencing trace reads (Hsiau et al 2019). ICE analysis shows that targeting AAVS1…”

Section: Mad7 Induces Double-strand Breaks In Human Cellsmentioning

confidence: 99%

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

Wierson

Simone

WareJoncas

et al. 2019

Preprint

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) effector proteins enable the direction of DNA double-strand breaks at defined loci based on a variable length RNA guide specific to each effector.The guides are generally similar in size and form, consisting of a ~20 base sequence homologous to the DNA target and a secondary structure used by the effector for guide/nuclease recognition. However, the effector proteins vary in size, DNA binding kinetics, nucleic acid hydrolyzing activities, and Protospacer Adjacent Motif (PAM) requirements. Recently, a Cas12a family member protein named Mad7 was identified that is most similar to the Cas12a from Acidaminococcus sp. Here, we report for the first time Mad7 activity in zebrafish and human cells. We utilize a fluorescent reporter system to demonstrate CRISPR/Mad7 elicits strand annealing mediated DNA repair more efficiently than CRISPR/Cas9. Finally, we use CRISPR/Mad7 with our previously reported gene targeting method GeneWeld in order to integrate reporter alleles in both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR/Cas system, increasing the flexibility researchers have in applying genome engineering technologies.

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Inference of CRISPR Edits from Sanger Trace Data

Cited by 322 publications

References 5 publications

The use of CRISPR for variant specificity in the genetic diagnosis of primary immunodeficiency disease (PID)

The use of CRISPR for variant specificity in the genetic diagnosis of primary immunodeficiency disease (PID)

A simple and efficient workflow for generation of knock‐in mutations in Jurkat T cells using CRISPR/Cas9

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

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