Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
The present invention generally relates to libraries, kits, methods, applications and screens used in functional genomics that focus on gene function in a cell and that may use vector systems and other aspects related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems and components thereof. The present invention also relates to rules for making potent single guide RNAs (sgRNAs) for use in CRISPR-Cas systems. Provided are genomic libraries and genome wide libraries, kits, methods of knocking out in parallel every gene in the genome, methods of selecting individual cell knock outs that survive under a selective pressure, methods of identifying the genetic basis of one or more medical symptoms exhibited by a patient, and methods for designing a genome-scale sgRNA library.
1. A genome-wide screening method, comprising knocking out in parallel a plurality of genes in a genome by a method which comprises:
(a) selecting a plurality of guide sequences from a library of candidate guide sequences, each guide sequence targeting a DNA molecule encoding a gene product, wherein a guide sequence is selected if the candidate guide sequence satisfies the following rules:
(i) binding affinity between the candidate guide sequence and a CRISPR-Cas protein is higher than binding affinity between a reference guide sequence in the library and the CRISPR-Cas protein,
(ii) none of the last four nucleotides of the candidate guide sequence is a pyrimidine, and
(iii) the candidate guide sequence has a % GC nucleotide content between 20% and 80%; and
(b) introducing into a population of cells a composition comprising:
(i) a plurality of guide polynucleotide sequences, each comprising a guide sequence from (a), whereby different guide polynucleotide sequences are introduced into different cells of the population; and
(ii) a Type II CRISPR-Cas protein or a polynucleotide sequence encoding a Type II CRISPR-Cas protein,
wherein each guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a genomic locus of the DNA molecule encoding the gene product,
wherein the CRISPR complex comprises the CRISPR-Cas protein complexed with a guide sequence that is hybridized to the target sequence, and
wherein the CRISPR-Cas protein cleaves the genomic locus of the DNA molecule encoding the gene product, whereby each cell in the population of cells has a unique gene knocked out in parallel.
2. The method of claim 1 , wherein the selection of the guide sequences further comprises predicting the efficacy of a guide sequence based on one or more of:
targeting early constitutive exons of coding genes, or
targeting of a non-transcribed DNA strand.
3. The method of claim 1 , wherein the cell is a eukaryotic cell.
4. The method of claim 3 , wherein the eukaryotic cell is a human cell, animal cell, or plant cell.
5. The method of claim 1 , wherein the CRISPR-Cas system guide polynucleotide sequences and the CRISPR-Cas protein are comprised in one or more vectors.
6. The method of claim 5 , wherein the one or more vectors is a lentivirus, an adenovirus or an AAV vector.
7. The method of claim 1 , where the CRISPR-Cas system guide polynucleotide sequences and the CRISPR-Cas protein are comprised in one vector.
8. The method of claim 7 , wherein the one vector is a lentivirus, an adenovirus or an AAV vector.
9. The method of claim 1 , wherein the targeting is of about 100 or more sequences.
10. The method of claim 1 , wherein the CRISPR-Cas protein is a Cas9.
11. The method of claim 10 , wherein the Cas9 protein is Streptococcus pyogenes Cas9 or Staphylococcus aureus Cas9.
12. The method of claim 1 , wherein the targeting is of about 1000 or more sequences.
13. The method of claim 1 , wherein the targeting is of about 20,000 or more sequences.
14. The method of claim 1 , wherein the targeting is of the entire genome.
15. The method of claim 1 , further comprising ranking the candidate guide sequences based on off-target scores and selecting guide sequences with the lowest scores.
16. The method of claim 1 , wherein each cell has introduced a single CRISPR-Cas system guide polynucleotide sequence.
17. The method of claim 1 , wherein the cells are transduced with a multiplicity of infection (MOI) of 0.3-0.75.
18. The method of claim 1 , comprising selecting guide sequences that target early constitutive exons of coding genes and/or selecting guide sequences that target non-transcribed DNA strands.
19. The method of claim 1 , further comprising selecting guide sequences that do not comprise a thymine at any one of the last four nucleotides of the guide sequences.