Cloning-free CRISPR

Abstract

We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%-4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.

Graphical abstract

Figure 1

Simplified, Efficient Genome Editing Using scCRISPR (A) Schematic shows the scCRISPR/Cas9 process that occurs inside target cells. (B) Histograms show flow cytometric GFP fluorescence (x axis) in Hist1h3a mouse ESCs (left) after electroporation with Cas9 and plasmid sgRNA (second from left), sgPal1 plasmid alone (third from left), and sgPal1 plasmid and sgGFP homology fragment with standard-length arms (fourth from left). (C) Fluorescence microscopy shows GFP fluorescence in Hist1h3a-GFP mouse ESCs (left) after targeting with Cas9 and plasmid sgRNA (second from left) and sgPal1 plasmid and sgGFP homology fragment (third from left). (D) Histograms show flow cytometric GFP fluorescence (x axis) in Hist1h3a-GFP knockin mouse ESCs after electroporation with Cas9 and (from left to right) sgPal1, sgPal7, and sgPal8 plasmids together with a long sgGFP homology fragment. (E) MiSeq plasmid copy numbers per cell of sgPal1, sgGFP, and the three most frequently mismatched sgGFP species 96 hr after co-electroporation of mouse ESCs are shown. (F) Multiplexed mutation of GFP (x axis) and dsRed (y axis) in Hist1h3a-GFP Rosa26-dsRed mouse ESCs (left) after co-introduction of Cas9, sgPal1 plasmid, and sgGFP and sgDsRed long-armed homology fragments (right) is shown. See also Figure S1.

Figure 2

Efficient, Cloning-Free Knockin Transgenesis Using PCR-Amplified Homology Arms (A) Flow cytometric analysis shows efficient generation of Hist1h3a-GFP knockin mouse ESCs (y axis) using a PCR-amplified GFP fragment with 80 bp Hist1h3a homology arms and plasmid-based sgRNA (left) or scCRISPR sgRNA (right). (B) Fluorescence microscopy shows Hist1h3a-GFP mouse ESCs generated through scCRISPR-based knockin. (C) Flow cytometric analysis shows efficient generation of HIST1H2BJ-GFP knockin HUES2 human ESCs (y axis) with PCR-amplified homology arms and plasmid-based sgRNA (second from left) or scCRISPR-based sgRNA (right). Untargeted human ESC fluorescence is shown for comparison (left). (D) Fluorescence microscopy shows HIST1H2BJ-GFP human ESCs generated through plasmid-based (left) or scCRISPR-based (right) knockin. (E and F) Flow cytometric analysis shows that a cloning-free approach introducing a gBlock sgRNA and a PCR-amplified homology fragment leads to even more efficient generation of Hist1h3a-GFP knockin mouse ESCs (E) and HIST1H2BJ-GFP human ESCs (F). See also Figure S2.

Figure 3

An scCRISPR-Based NHEJ Gene Knockout Screen Improves HR Efficiency 3-Fold Hist1h3a-GFP knockin efficiency (y axis) is shown for 13 scCRISPR-generated bulk knockout lines of genes reported to play a role in NHEJ (x axis). Values and SDs are averaged from three independent biological experiments. A clonal double knockout line for Prkdc and Lig4 (red) exhibits 3-fold more efficient HR than wild-type mouse ESCs (dotted line).