Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array

Abstract

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

Figure 1. Cpf1 mediated processing of pre-crRNA is independent of DNA cleavage

(a) Schematic of pre-crRNA processing for Cas9 and Cpf1. Cleavage sites indicated with red triangles. (b) In vitro processing of FnCpf1 pre-crRNA transcript (80 nM) with purified AsCpf1 or LbCpf1 protein (~320 nM), cropped gel image. (c) RNAseq analysis of FnCpf1 pre-crRNA cleavage products, as shown in (b). A high fraction of sequence reads smaller than 65nt are cleavage products of spacers flanked by DR sequences, cropped gel images. (d) Pre-crRNA (top) and DNA cleavage (bottom) mediated by AsCpf1 point mutants. H800A, K809A, K860A, F864A, and R790A fail to process precrRNA but retain DNA cleavage activity in vitro. 330 nM pre-crRNA was cleaved with 500 nM Cpf1 in 15 min and 25 nM DNA was cleaved with 165 nM Cpf1 in 30 min. (e) Indel frequencies mediated by AsCpf1H800A are comparable to wt AsCpf1, bars are mean of 3 technical replicates from one experiment, error bars are SEM. (Student t-test; ns = not significant; ** = p-value 0.003). (f) Schematic of lenti-Cpf1 construct with the U6::DR cassette in different orientations (top and middle), (+)-strand lenti RNA with recognizable DRs are susceptible to Cpf1 mediated degradation, preventing functional virion formation. Schematic of lenti-AsCpf1 (pY108) construct (bottom). (g) Indel frequencies analyzed by SURVEYOR nuclease assay after puromycin selection 10 days after transduction with lenti-AsCpf1 in HEK cells, bars are mean of 2 or 3 individual infections, error bars are SEM. U6, Pol III promoter; CMV, cytomegalovirus promoter; NLS, nuclear localization signal; HA, hemagglutinin tag; DR, direct repeat sequence; P2A, porcine teschovirus-1 2A self-cleaving peptide; LTR, long terminal repeat; WPRE, Woodchuck Hepatitis virus posttranscriptional regulatory element.

Figure 2. Cpf1-mediated multiplex gene editing in mammalian cells and mouse brain

(a) Schematic of multiplex gene editing with AsCpf1, using a single plasmid approach. (b) Genome editing at four different genomic loci mediated by AsCpf1 with different versions of artificial CRISPR arrays (array-1, crRNAs in their mature form (19nt DR with 23nt guide); array-2, crRNAs are in an intermediate form (19nt DR with 30nt guide); array-3 crRNAs are in their unprocessed form (35nt DR with 30nt guides)). Indels were analyzed by SURVEYOR nuclease assay 3 days post transfection; bars are mean of two individual experiments with 3 to 5 technical replicates, error bars are SEM. (c) Small RNAseq reads from HEK cells transfected with AsCpf1 and array-1 show fragments corresponding to mature crRNA for each of the four guides. (d) Schematic for analysis of indel events in clonal colonies 48 hours after transient transfection. (e) Quantification of indel events measured by NGS in clonal colonies from HEK cells transiently transfected with pooled single guide plasmids or plasmid carrying array-1. Colonies were expanded for 10 days after sorting. Each column represents one clonal colony; blue rectangles indicate target genes with all alleles edited. (f) Schematic of AAV vector design for multiplex gene editing. Bottom: grey rectangles, direct repeat; diamonds, spacer (red: Mecp2, orange: Nlgn3, green: Drd1). (g) Immunostaining of dorsal DG 4 weeks after stereotactic AAV injection (Representative image of n = 4 mice). Brain sections were co-stained with anti-HA (red), anti-GFP (green) and anti-NeuN (magenta) antibodies. Nuclei were labeled with DAPI (blue). Scale bar: 100 um. (h) Western blot analysis of DG expressing HA-AsCpf1 and GFP-KASH (Representative blot from n = 4 mice). (i) Fraction of mono- and biallelic modifications of autosomal gene Drd1 is shown (Mecp2 and Nlgn3: x-chromosomal). (j) Analysis of multiplexing efficiency in individual cells. ITR, inverted terminal repeat; spA, synthetic polyadenylation signal; hSyn1, human synapsin 1 promoter; ANC1, Syne Homology nuclear transmembrane domain; hGH pA, human growth hormone polyadenylation signal;